Compositions and Methods for Identifying and Targeting Cancer Cells of Alimentary Canal Origin

ABSTRACT

Screening and diagnostic reagents, kits and methods for primary and/or metastatic stomach or esophageal cancer are disclosed. Compositions for and methods of imaging and treating primary and/or metastatic stomach or esophageal cancer are disclosed. Vaccines compositions and methods of for treating and preventing primary and/or metastatic stomach or esophageal cancer are disclosed.

CROSS REFERENCE TO RELATED PATENT APPLICATIONS

This application claims priority to U.S. Provisional Application No.60/192,229 filed Mar. 27, 2000, which is incorporated herein byreference.

This application is also related to U.S. Pat. No. 5,518,888, issued May21, 1996, U.S. Pat. No. 5,601,990 issued Feb. 11, 1997, U.S. Pat. No.6,060,037 issued Apr. 26, 2000, U.S. Pat. No. 5,962,220 issued Oct. 5,1999, and U.S. Pat. No. 5,879,656 issued Mar. 9, 1999, which are eachincorporated herein by reference and U.S. patent application Ser. No.09/180,237 filed Mar. 12, 1997, which is incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to in vitro diagnostic methods fordetecting cancer cells of the alimentary canal, particularly primary andmetastatic stomach and esophageal cancer, and to kits and reagents forperforming such methods. The present invention relates to compounds andmethods for in vivo imaging and treatment of tumors originating from thealimentary canal, particularly primary and metastatic stomach andesophageal tumors. The present invention relates to methods andcompositions for making and using targeted gene therapy, antisense anddrug compositions. The present invention relates to prophylactic andtherapeutic vaccines against cancer cells of the alimentary canal,particularly primary and metastatic stomach and esophageal cancer andcompositions and methods of making and using the same.

BACKGROUND OF THE INVENTION

There is a need for reagents, kits and methods for screening, diagnosingand monitoring individuals with cancer originating from the alimentarycanal, particularly primary and metastatic stomach and esophagealcancer. There is a need for reagents, kits and methods for identifyingand confirming that a cancer of unknown origin is originating from thealimentary canal and for analyzing tissue and cancer samples to identifyand confirm cancer originating from the alimentary canal and todetermine the level of migration of such cancer cells. There is a needfor compositions which can specifically target stomach and esophagealcancer cells. There is a need for imaging agents which can specificallybind to stomach and esophageal cancer cells. There is a need forimproved methods of imaging stomach and esophageal cancer cells. Thereis a need for therapeutic agents which can specifically bind to stomachand esophageal cancer cells. There is a need for improved methods oftreating individuals who are suspected of suffering from primary and/ormetastatic stomach or esophageal cancer. There is a need for vaccinecomposition to treat stomach and esophageal cancer. There is a need forvaccine composition to treat and prevent stomach and esophageal cancer.There is a need for therapeutic agents which can specifically delivergene therapeutics, antisense compounds and other drugs to stomach andesophageal cancer cells.

SUMMARY OF THE INVENTION

The invention further relates to in vitro methods of determining whetheror not an individual has cancer originating from the alimentary canal,particularly primary and metastatic stomach and esophageal cancer. Thepresent invention relates to in vitro methods of examining samples ofnon-colorectal tissue and body fluids from an individual to determinewhether or not GCC, which is expressed by normal colon cells and bycolorectal, stomach and esophageal tumor cells, is being expressed bycells in samples other than colon. The presence of GCC protein or of theGCC gene transcript in samples outside the colorectal track isindicative of expression of GCC and is evidence that the individual maybe suffering from metastasized colon cancer or primary or metastaticstomach and/or esophageal cancer. In patients suspected of sufferingfrom colorectal cancer, the presence of GCC protein or of the GCC genetranscript in samples outside the colorectal track is supportive of theconclusion that the individual is suffering from metastatic colorectalcancer. The diagnosis of metastatic colorectal cancer may be made orconfirmed. In patients suspected of suffering from stomach or esophagealcancer, the presence of GCC protein or of the GCC gene transcript insamples outside the colorectal track is supportive of the conclusionthat the individual is suffering from primary and/or metastatic stomachor esophageal cancer. The diagnosis of primary and/or metastatic stomachor esophageal cancer may be made or confirmed.

The invention further relates to in vitro methods of determining whetheror not tumor cells suspected of being stomach or esophageal cancer arestomach or esophageal in origin. The present invention relates to invitro methods of diagnosing whether or not an individual suspected ofsuffering from stomach or esophageal cancer is suffering from stomach oresophageal cancer. The present invention relates to in vitro methods ofexamining samples of tumors from an individual to determine whether ornot GCC protein, which is expressed by colorectal, stomach or esophagealtumor cells, is being expressed by the tumor cells. The presence of aGCC protein or of the GCC gene transcript in a sample from a patientsuspected of having stomach or esophageal cancer is indicative ofexpression of GCC and evidence that the individual may be suffering fromstomach or esophageal cancer. In tumors which are suspected of beingstomach or esophageal tumors, the presence of a GCC protein or of theGCC gene transcript supports the conclusion that the tumors are ofstomach or esophageal cancer and the diagnosis of stomach or esophagealcancer.

The invention further relates to in vitro kits for practicing themethods of the invention and to reagents and compositions useful ascomponents in such in vitro kits of the invention.

The invention further relates to a method of imaging primary andmetastatic stomach and esophageal tumors and to methods of treating anindividual suspected of suffering from primary and metastatic stomachand esophageal tumors comprising the steps of administering to saidindividual a pharmaceutical compositions according to the invention,wherein the compositions or conjugated compounds are present in anamount effective for therapeutic or diagnostic use in humans sufferingfrom primary and/or metastatic stomach or esophageal tumors.

The invention further relates to a method of delivering an active agentto primary and metastatic stomach and esophageal tumor cells comprisingthe steps of administering to an individual who has primary and/ormetastatic stomach or esophageal tumors, a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier or diluent, and anunconjugated compositions that comprises a liposome that includes GCCligands on its surface and an active component encapsulated therein.

The invention further relates to killed or inactivated stomach oresophageal tumor cells that comprise a protein comprising at least oneepitope of a GCC protein; and to vaccines comprising the same. In someembodiments, the killed or inactivated cells or particles comprise a GCCprotein. In some embodiments, the killed or inactivated cells orparticles are haptenized.

The invention further relates to methods of treating individualssuffering from stomach or esophageal cancer and to methods of treatingindividuals susceptible stomach or esophageal cancer. The method of thepresent invention provides administering to such individuals aneffective amount of such vaccines. The invention further relates to theuse of such vaccines as immunotherapeutics.

DESCRIPTION OF PREFERRED EMBODIMENTS Definitions

As used herein, the term “GCC” is meant to refer to the cellular proteinexpressed by normal colorectal cells, as well as primary andmetastasized colorectal, stomach and esophageal cancer cells. In normalindividuals, GCC is found exclusively in cells of intestine, inparticular in cells in the duodenum, small intestine (jejunum andileum), the large intestine, colon (cecum, ascending colon, transversecolon, descending colon and sigmoid colon) and rectum.

As used herein, the term “functional fragment” as used in the term“functional fragment of a GCC gene transcript” is meant to refer tofragments of GCC gene transcript which are functional with respect tonucleic acid molecules with full length sequences. For example, afunctional fragment may be useful as an oligonucleotide or nucleic acidprobe, a primer, an antisense oligonucleotide or nucleic acid moleculeor a coding sequence. The nucleotide sequence encoding human GCC proteinis disclosed in F. J. Sauvage et al. 1991 J. Biol. Chem.266:17912-17918, each of which is incorporated herein by reference.

As used herein, the term “functional fragment” as used in the term“functional fragment of a GCC protein” is meant to fragments of GCCprotein which function in the same manner as GCC protein with fulllength sequences. For example, an immunogenically functional fragment ofa GCC protein comprises an epitope recognized by an anti-GCC antibody. Aligand-binding functional fragment of GCC comprises a sequence whichforms a structure that can bind to a ligand which recognizes and bindsto GCC protein.

As used herein, the term “epitope recognized by an anti-GCC proteinantibody” refers to those epitopes specifically recognized by ananti-GCC protein antibody.

As used herein, the term “antibody” is meant to refer to complete,intact antibodies, and Fab fragments and F(ab)₂ fragments thereof.Complete, intact antibodies include monoclonal antibodies such as murinemonoclonal antibodies, chimeric antibodies and humanized antibodies.

As used herein, the term “GCC ligand” is meant to refer to compoundswhich specifically bind to a GCC protein. Antibodies that bind to GCCare GCC ligands. A GCC ligand may be a protein, peptide or anon-peptide.

As used herein, the term “active agent” is meant to refer to compoundsthat are therapeutic agents or imaging agents.

As used herein, the term “radiostable” is meant to refer to compoundswhich do not undergo radioactive decay; i.e. compounds which are notradioactive.

As used herein, the term “therapeutic agent” is meant to refer tochemotherapeutics, toxins, radiotherapeutics, targeting agents orradiosensitizing agents. As used herein, the term “chemotherapeutic” ismeant to refer to compounds that, when contacted with and/orincorporated into a cell, produce an effect on the cell includingcausing the death of the cell, inhibiting cell division or inducingdifferentiation.

As used herein, the term “toxin” is meant to refer to compounds that,when contacted with and/or incorporated into a cell, produce the deathof the cell.

As used herein, the term “radiotherapeutic” is meant to refer toradionuclides which when contacted with and/or incorporated into a cell,produce the death of the cell.

As used herein, the term “targeting agent” is meant to refer compoundswhich can be bound by and or react with other compounds. Targetingagents may be used to deliver chemotherapeutics, toxins, enzymes,radiotherapeutics, antibodies or imaging agents to cells that havetargeting agents associated with them and/or to convert or otherwisetransform or enhance co-administered active agents. A targeting agentmay include a moiety that constitutes a first agent that is localized tothe cell which when contacted with a second agent either is converted toa third agent which has a desired activity or causes the conversion ofthe second agent into an agent with a desired activity.

The result is the localized agent facilitates exposure of an agent witha desired activity to the cancer cell.

As used herein, the term “radiosensitizing agent” is meant to refer toagents which increase the susceptibility of cells to the damagingeffects of ionizing radiation. A radiosensitizing agent permits lowerdoses of radiation to be administered and still provide atherapeutically effective dose.

As used herein, the term “imaging agent” is meant to refer to compoundswhich can be detected.

As used herein, the term “GCC binding moiety” is meant to refer to theportion of a conjugated compound that constitutes an GCC ligand.

As used herein, the term “active moiety” is meant to refer to theportion of a conjugated compound that constitutes an active agent.

As used herein, the terms “conjugated compound” and “conjugatedcomposition” are used interchangeably and meant to refer to a compoundwhich comprises a GCC binding moiety and an active moiety and which iscapable of binding to GCC. Conjugated compounds according to the presentinvention comprise a portion which constitutes an GCC ligand and aportion which constitutes an active agent. Thus, conjugated compoundsaccording to the present invention are capable of specifically bindingto the GCC and include a portion which is a therapeutic agent or imagingagent. Conjugated compositions may comprise crosslinkers and/ormolecules that serve as spacers between the moieties.

As used herein, the terms “crosslinker”, “crosslinking agent”,“conjugating agent”, “coupling agent”, “condensation reagent” and“bifunctional crosslinker” are used interchangeably and are meant torefer to molecular groups which are used to attach the GCC ligand andthe active agent to thus form the conjugated compound.

As used herein, the term “colorectal cancer” is meant to include thewell-accepted medical definition that defines colorectal cancer as amedical condition characterized by cancer of cells of the intestinaltract below the small intestine (i.e. the large intestine (colon),including the cecum, ascending colon, transverse colon, descendingcolon, and sigmoid colon, and rectum). Additionally, as used herein, theterm “colorectal cancer” is meant to further include medical conditionswhich are characterized by cancer of cells of the duodenum and smallintestine (jejunum and ileum). The definition of colorectal cancer usedherein is more expansive than the common medical definition but isprovided as such since the cells of the duodenum and small intestinealso contain GCC.

As used herein, the term “stomach cancer” is meant to include thewell-accepted medical definition that defines stomach cancer as amedical condition characterized by cancer of cells of the stomach.

As used herein, the term “esophageal cancer” is meant to include thewell-accepted medical definition that defines esophageal cancer as amedical condition characterized by cancer of cells of the esophagus.

As used herein, the term “metastasis” is meant to refer to the processin which cancer cells originating in one organ or part of the bodyrelocate to another part of the body and continue to replicate.Metastasized cells subsequently form tumors which may furthermetastasize. Metastasis thus refers to the spread of cancer from thepart of the body where it originally occurs to other parts of the body.

As used herein, the term “metastasized colorectal cancer cells” is meantto refer to colorectal cancer cells which have metastasized.Metastasized colorectal cancer cells localized in a part of the bodyother than the duodenum, small intestine (jejunum and ileum), largeintestine (colon), including the cecum, ascending colon, transversecolon, descending colon, and sigmoid colon, and rectum.

As used herein, the term “metastasized stomach cancer cells” is meant torefer to stomach cancer cells which have metastasized. Metastasizedstomach cancer cells localized in a part of the body other than thestomach.

As used herein, the term “metastasized esophageal cancer cells” is meantto refer to colorectal cancer cells which have metastasized.Metastasized esophageal cancer cells localized in a part of the bodyother than the esophagus.

As used herein, the term “non-colorectal sample” and “extra-intestinalsample” are used interchangeably and meant to refer to a sample oftissue or body fluid from a source other than colorectal tissue. In somepreferred embodiments, the non-colorectal sample is a sample of tissuesuch as lymph nodes. In some preferred embodiments, the non-colorectalsample is a sample of extra-intestinal tissue which is an adenocarcinomaof unconfirmed origin. In some preferred embodiments, the non-colorectalsample is a blood sample.

As used herein, “an individual suffering from an adenocarcinoma ofunconfirmed origin” is meant to refer to an individual who has a tumorin which the origin has not been definitively identified.

As used herein, “an individual is suspected of being susceptible tostomach or esophageal cancer” is meant to refer to an individual who isat a particular risk of developing stomach or esophageal cancer.Examples of individuals at a particular risk of developing stomach oresophageal cancer are those whose family medical history indicates aboveaverage incidence of stomach or esophageal cancer among family membersand/or those who have already developed stomach or esophageal cancer andhave been effectively treated who therefore face a risk of relapse andrecurrence.

As used herein, the term “antisense composition” and “antisensemolecules” are used interchangeably and are meant to refer to compoundsthat regulate transcription or translation by hybridizing to DNA or RNAand inhibiting and/or preventing transcription or translation fromtaking place. Antisense molecules include nucleic acid molecules andderivatives and analogs thereof. Antisense molecules hybridize to DNA orRNA in the same manner as complementary nucleotide sequences doregardless of whether or not the antisense molecule is a nucleic acidmolecule or a derivative or analog. Antisense molecules may inhibit orprevent transcription or translation of genes whose expression is linkedto cancer.

As used herein, the term “GCC immunogen” is meant to refer to GCCprotein or a fragment thereof or a protein that comprises the same or ahaptenized product thereof, cells and particles which display at leastone GCC epitope, and haptenized cells and haptenized particles whichdisplay at least one GCC epitope.

As used herein, the term “recombinant expression vector” is meant torefer to a plasmid, phage, viral particle or other vector which, whenintroduced into an appropriate host, contains the necessary geneticelements to direct expression of the coding sequence that encodes theprotein. The coding sequence is operably linked to the necessaryregulatory sequences. Expression vectors are well known and readilyavailable. Examples of expression vectors include plasmids, phages,viral vectors and other nucleic acid molecules or nucleic acid moleculecontaining vehicles useful to transform host cells and facilitateexpression of coding sequences.

As used herein, the term “illegitimate transcription” is meant to referto the low level or background expression of tissue-specific genes incells from other tissues.

The phenomenon of illegitimate transcription thus provides copies ofmRNA for a tissue specific transcript in other tissues. If detectiontechniques used to detect gene expression are sufficiently sensitive todetect illegitimate transcription, the expression level of thetranscript in negative samples due to illegitimate transcription must bediscounted using controls and/or quantitative assays and/or other meansto eliminate the incidence of false positive due to illegitimatetranscription. Alternatively, detection of evidence of GCC geneexpression in sample is achieved without detecting GCC gene transcriptpresent due to illegitimate transcription. This is accomplished usingtechniques which are not sufficiently sensitive to detect the GCC genetranscript present due to illegitimate transcription which is present asbackground.

U.S. Pat. No. 5,518,888, issued May 21, 1996, U.S. Pat. No. 5,601,990issued Feb. 11, 1997, U.S. Pat. No. 6,060,037 issued Apr. 26, 2000, U.S.Pat. No. 5,962,220 issued Oct. 5, 1999, U.S. Pat. No. 5,879,656 issuedMar. 9, 1999, and U.S. patent application Ser. No. 09/180,237 filed Mar.12, 1997, relate to targeting ST receptors to treat, image, detect andvaccinate against metastasized colorectal cancer. It has now beendiscovered that primary and metastatic stomach and esophageal cancercells express ST receptors (GCC). Accordingly, the compositions andmethods described in the above-listed patents and application can beused to treat, image, detect and vaccinate against primary andmetastatic stomach and esophageal cancer. The present invention adaptsthe earlier invention as related to metastasized colorectal cancer totreat, image, detect and vaccinate against primary and metastaticstomach and esophageal cancer.

GCC

Carcinomas derived from the colorectal cells, stomach or esophagusexpress GCC. The expression of GCC by such tumors enables this proteinand its mRNA to be a specific biomarker for the presence of cancer cellsin extra-intestinal tissues and blood. Indeed, this characteristicpermits the detection of GCC mRNA by RT-PCR analysis to be a diagnostictest to stage patients with colorectal, stomach or esophageal cancer andfollow patients after surgery for evidence of recurrent disease in theirblood as well as to detect colorectal, stomach and esophageal cancers.Further, the GCC may be targeted with a ligand conjugated to an activeagent in order to deliver the active agent to tumor cells in vivo.

U.S. Pat. No. 5,518,888 issued May 21, 1996 to Waldman, PCT applicationPCT/US94/12232 filed Oct. 26, 1994, U.S. application Ser. No. 08/467,920filed Jun. 6, 1995, and U.S. application Ser. No. 08/583,447 filed Jan.5, 1996, which are each incorporated herein by reference, disclose thatmetastasized colorectal tumors can be targeted for delivery of activecompounds by targeting ST receptors (also referred to as guanylincyclase C or GCC). The presence of ST receptors on cells outside of theintestinal tract as a marker for colorectal cancer allows for thescreening, identification and treatment of individuals with metastasizedcolorectal tumors. ST receptors may also be used to target delivery ofgene therapeutics and antisense compounds to colorectal cells.

U.S. Pat. No. 5,601,990 issued Feb. 11, 1997 to Waldman, PCT applicationPCT/US94/12232 filed Oct. 26, 1994, and PCT application PCT/US97/07467filed May 2, 1997, which are each incorporated herein by reference,disclose that detection of evidence of expression of ST receptors insamples of tissue and body fluid from outside the intestinal trackindicate metastasized colorectal cancer.

PCT application PCT/US97/07565 filed May 2, 1997, which is incorporatedherein by reference, disclose that immunogens with epitopes that can betargeted by antibodies that react with ST receptors can be used invaccines compositions useful as prophylactic and therapeuticanti-metastatic colorectal cancer compositions.

It has been discovered that, in addition to normal colon cells, toprimary and to metastasized colon, stomach and esophageal carcinomacells also express GCC. Normal stomach and esophageal cells do notexpress GCC. Thus, the present invention provides the use of GCC as aspecific molecular diagnostic marker for the diagnosis, staging, andpost-operative surveillance of patients with primary and metastasizedstomach and esophageal cancer.

Detection of the expression of GCC employing molecular techniques,including, but not limited to, RT-PCR, can be employed to diagnose andstage patients, follow the development of recurrence after surgeryand/or remission, and, potentially, screen normal people for thedevelopment of colorectal, stomach or esophageal cancer.

GCC is unique in that it is only expressed in normal intestinal cells.Mucosal cells lining the intestine are joined together by tightjunctions which form a barrier against the passage of intestinalcontents into the blood stream and components of the blood stream intothe intestinal lumen. Therefore, the apical location of cells expressingGCC results in the isolation of such cells from the circulatory systemso that they may be considered to exist separate from the rest of thebody; essentially the “outside” of the body. Therefore, the rest of thebody is considered “outside” the intestinal tract. Compositionsadministered “outside” the intestinal tract are maintained apart andsegregated from the only cells which normally express GCC. Conversely,tissue samples taken from tissue outside of the intestinal tract do notnormally contain cells which express GCC.

In individuals suffering from colorectal cancer, the cancer cells areoften derived from cells that produce and display the GCC and thesecancer cells continue to produce GCC. It has been observed that GCC isexpressed by colorectal cancer cells. Likewise, GCC is expressed bystomach and esophageal cancer cells.

The expression of GCC by colorectal tumor cells provides a detectabletarget for in vitro screening, monitoring and staging as well as atarget for in vivo delivery of conjugated compositions that compriseactive agents for the imaging and treatment. GCC can also serve astargets for vaccines which may be used to protect against metastasizedcolorectal cancer or to treat individuals with metastasized colorectalcancer.

The expression of GCC by stomach and esophageal tumor cells provides adetectable target for in vitro screening, monitoring and staging as wellas a target for in vivo delivery of conjugated compositions thatcomprise active agents for the imaging and treatment. GCC can also serveas targets for vaccines which may be used to protect against primary andmetastatic stomach and esophageal cancer or to treat individuals withprimary and metastatic stomach and esophageal cancer.

In Vitro Diagnostics

According to some embodiments of the invention, compositions, kits andin vitro methods are provided for screening, diagnosing and analyzingpatients and patient samples to detect evidence of GCC expression bycells outside of the intestinal tract wherein the expression of GCC maybe suggestive of metastasized colorectal cancer or primary or metastaticstomach or esophageal cancer. In patients suspected of having primary ormetastatic stomach or esophageal cancer evidence of GCC expression bycells outside of the intestinal tract is indicative of primary ormetastatic stomach or esophageal cancer and can be used in thediagnosis, monitoring and staging of such patients. Furthermore, thepresent invention relates to methods, compositions and kits useful inthe in vitro screening, and analysis of patient and patient samples todetect evidence of GCC expression by tumor cells outside of theintestinal tract wherein the presence of cells that express GCC suggestsor confirms that a tumor is of colorectal or stomach or esophagealcancer origin. In an additional aspect of the invention, compositions,kits and methods are provided which are useful to visualize primary ormetastatic stomach or esophageal cancer cells.

In vitro screening and diagnostic compositions, methods and kits can beused in the monitoring of individuals who are in high risk groups forstomach or esophageal cancer such as those who have been diagnosed withlocalized disease and/or metastasized disease and/or those who aregenetically linked to the disease. In vitro screening and diagnosticcompositions, methods and kits can be used in the monitoring ofindividuals who are undergoing and/or have been treated for primarystomach or esophageal cancer to determine if the cancer hasmetastasized. In vitro screening and diagnostic compositions, methodsand kits can be used in the monitoring of individuals who are undergoingand/or have been treated for stomach or esophageal cancer to determineif the cancer has been eliminated. In vitro screening and diagnosticcompositions, methods and kits can be used in the monitoring ofindividuals who are otherwise susceptible, i.e. individuals who havebeen identified as genetically predisposed such as by genetic screeningand/or family histories. Advancements in the understanding of geneticsand developments in technology as well as epidemiology allow for thedetermination of probability and risk assessment an individual has fordeveloping stomach or esophageal cancer. Using family health historiesand/or genetic screening, it is possible to estimate the probabilitythat a particular individual has for developing certain types of cancerincluding stomach or esophageal cancer. Those individuals that have beenidentified as being predisposed to developing a particular form ofcancer can be monitored or screened to detect evidence of stomach oresophageal cancer. Upon discovery of such evidence, early treatment canbe undertaken to combat the disease. Accordingly, individuals who are atrisk for developing stomach or esophageal cancer may be identified andsamples may be isolated form such individuals.

The invention is particularly useful for monitoring individuals who havebeen identified as having family medical histories which includerelatives who have suffered from stomach or esophageal cancer. Likewise,the invention is particularly useful to monitor individuals who havebeen diagnosed as having stomach or esophageal cancer and, particularlythose who have been treated and had tumors removed and/or are otherwiseexperiencing remission including those who have been treated for stomachor esophageal cancer.

In vitro screening and diagnostic compositions, methods and kits can beused in the analysis of tumors. Expression of GCC is a marker for celltype and suggests the origin of adenocarcinoma of unconfirmed originsuspected of being gastric or esophageal in origin may be stomach oresophageal tumors. Detection of GCC expression can also be used toassist in an initial diagnosis of stomach or esophageal cancer or toconfirm such diagnosis. Tumors believed to be stomach or esophageal inorigin can be confirmed as such using the compositions, methods and kitsof the invention.

In vitro screening and diagnostic compositions, kits and methods of theinvention can be used to analyze tissue samples from the stomach oresophagus to identify primary stomach or esophageal cancer.

According to the invention, compounds are provided which bind to GCCgene transcript or protein. Normal tissue in the body does not have GCCtranscript or protein except cells of the intestinal tract. Theexpression of GCC is a marker for cell type and is useful in theidentification of stomach or esophageal cancer in extra-intestinalsamples.

In some embodiments of the invention, non-colorectal tissue and fluidsamples or tumor samples may be screened to identify the presence orabsence of GCC protein. Techniques such as ELISA assays and Westernblots may be performed to determine whether GCC is present in a sample.

In some embodiments of the invention, non-colorectal tissue and fluidsamples or tumor samples may be screened to identify whether GCC arebeing expressed in cells outside of the colorectal tract by detectingthe presence or absence of GCC gene transcript. The presence of GCC genetranscript or cDNA generated therefrom can be determined usingtechniques such as PCR amplification, branched oligonucleotidetechnology, Northern Blots (mRNA), Southern Blots (cDNA), oroligonucleotide hybridization.

In some embodiments of the invention, cells of non-colorectal tissuesamples or tumor samples may be examined to identify the presence orabsence of GCC proteins.

Techniques such as immunohistochemistry blots may be performed on tissuesections to determine whether GCC are present in a sample.

In some embodiments of the invention, cells of non-colorectal tissuesamples or tumor samples may be examined to determine whether GCC arebeing expressed in cells outside of the colorectal track by detectingthe presence or absence of the GCC gene transcript. The presence of theGCC gene transcript or cDNA generated therefrom in cells from tissuesections can be determined using techniques such as in situhybridization.

The presence of GCC in non-colorectal tissue and fluid samples or oncells from non-colorectal tissue samples suggests possible stomach oresophageal cancer. The presence of GCC in a tumor sample or on tumorcells suggests that the tumor may be stomach or esophageal in origin.The presence of the GCC gene transcript in non-colorectal tissue andfluid samples or in cells from non-colorectal tissue samples suggestspossible stomach or esophageal cancer. The presence of the GCC genetranscript in tumor samples and tumor cells suggests that the tumor maybe stomach or esophageal in origin.

Samples may be obtained from resected tissue or biopsy materialincluding needle biopsy. Tissue section preparation for surgicalpathology may be frozen and prepared using standard techniques.Immunohistochemistry and in situ hybridization binding assays on tissuesections are performed in fixed cells. Extra-intestinal samples may behomogenized by standard techniques such as sonication, mechanicaldisruption or chemical lysis such as detergent lysis. It is alsocontemplated that tumor samples in body fluids such as blood, urine,lymph fluid, cerebral spinal fluid, amniotic fluid, vaginal fluid, semenand stool samples may also be screened to determine if such tumors arecolorectal, stomach or espophageal in origin.

Non-colorectal tissue samples may be obtained from any tissue exceptthose of the colorectal tract, i.e. the intestinal tract below the smallintestine (i.e. the large intestine (colon), including the cecum,ascending colon, transverse colon, descending colon, and sigmoid colon,and rectum) and additionally the duodenum and small intestine (jejunumand ileum). The normal cells of all tissue except those of thecolorectal tract do not express GCC. Thus if GCC protein or the GCC genetranscript are detected in non-colorectal samples, the possible presenceof colorectal, stomach or esophageal cancer cells is suggested. In somepreferred embodiments, the tissue samples are lymph nodes.

Tissue samples may be obtained by standard surgical techniques includinguse of biopsy needles. One skilled in the art would readily appreciatethe variety of test samples that may be examined for GCC and recognizemethods of obtaining tissue samples.

Tissue samples may be homogenized or otherwise prepared for screeningfor the presence of GCC by well known techniques such as sonication,mechanical disruption, chemical lysis such as detergent lysis orcombinations thereof.

Examples of body fluid samples include blood, urine, lymph fluid,cerebral spinal fluid, amniotic fluid, vaginal fluid and semen. In somepreferred embodiments, blood is used as a sample of body fluid. Cellsmay be isolated from fluid sample such as centrifugation. One skilled inthe art would readily appreciate the variety of test samples that may beexamined for GCC. Test samples may be obtained by such methods aswithdrawing fluid with a syringe or by a swab. One skilled in the artwould readily recognize other methods of obtaining test samples.

In an assay using a blood sample, the blood plasma may be separated fromthe blood cells. The blood plasma may be screened for GCC includingtruncated proteins which are released into the blood when one or moreGCC are cleaved from or sloughed off from tumor cells. In someembodiments, blood cell fractions are screened for the presence ofstomach or esophageal tumor cells. In some embodiments, lymphocytespresent in the blood cell fraction are screened by lysing the cells anddetecting the presence of GCC protein or the GCC gene transcript whichmay be present as a result of the presence of any stomach or esophagealtumor cells that may have been engulfed by the blood cell. In somepreferred embodiments, CD34+ cells are removed prior to isolation ofmRNA from samples using commercially available immuno-columns.

Aspects of the present invention include various methods of determiningwhether a sample contains cells that express GCC by nucleotidesequence-based molecular analysis to detect the GCC gene transcript.Several different methods are available for doing so including thoseusing Polymerase Chain Reaction (PCR) technology, branchedoligonucleotide technology, Northern blot technology, oligonucleotidehybridization technology, and in situ hybridization technology.

The invention relates to oligonucleotide probes and primers used in themethods of identifying the GCC gene transcript and to diagnostic kitswhich comprise such components.

The mRNA sequence-based methods for detect the GCC gene transcriptinclude but are not limited to polymerase chain reaction technology,branched oligonucleotide technology, Northern and Southern blottechnology, in situ hybridization technology and oligonucleotidehybridization technology.

The methods described herein are meant to exemplify how the presentinvention may be practiced and are not meant to limit the scope ofinvention. It is contemplated that other sequence-based methodology fordetecting the presence of the GCC gene transcript in non-colorectalsamples may be employed according to the invention.

A preferred method to detecting the GCC gene transcript in geneticmaterial derived from non-colorectal samples uses polymerase chainreaction (PCR) technology.

PCR technology is practiced routinely by those having ordinary skill inthe art and its uses in diagnostics are well known and accepted. Methodsfor practicing PCR technology are disclosed in “PCR Protocols: A Guideto Methods and Applications”, Innis, M. A., et al. Eds. Academic Press,Inc. San Diego, Calif. (1990) which is incorporated herein by reference.Applications of PCR technology are disclosed in “Polymerase ChainReaction” Erlich, H. A., et al., Eds. Cold Spring Harbor Press, ColdSpring Harbor, N.Y. (1989) which is incorporated herein by reference.U.S. Pat. No. 4,683,202, U.S. Pat. No. 4,683,195, U.S. Pat. No.4,965,188 and U.S. Pat. No. 5,075,216, which are each incorporatedherein by reference describe methods of performing PCR. PCR may beroutinely practiced using Perkin Elmer Cetus GENE AMP RNA PCR kit, PartNo. N808-0017.

PCR technology allows for the rapid generation of multiple copies of DNAsequences by providing 5′ and 3′ primers that hybridize to sequencespresent in an RNA or DNA molecule, and further providing freenucleotides and an enzyme which fills in the complementary bases to thenucleotide sequence between the primers with the free nucleotides toproduce a complementary strand of DNA. The enzyme will fill in thecomplementary sequences adjacent to the primers. If both the 5′ primerand 3′ primer hybridize to nucleotide sequences on the same smallfragment of nucleic acid, exponential amplification of a specificdouble-stranded size product results. If only a single primer hybridizesto the nucleic acid fragment, linear amplification producessingle-stranded products of variable length.

PCR primers can be designed routinely by those having ordinary skill inthe art using sequence information. The nucleotide sequence of the GCCgene transcript is set forth in SEQ ID NO:1. To perform this method, RNAis extracted from cells in a sample and tested or used to make cDNAusing well known methods and readily available starting materials. Thosehaving ordinary skill in the art can readily prepare PCR primers. A setof primers generally contains two primers. When performing PCR onextracted mRNA or cDNA generated therefrom, if the GCC gene transcriptor cDNA generated therefrom is present, multiple copies of the mRNA orcDNA will be made. If it is not present, PCR will not generate adiscrete detectable product. Primers are generally 8-50 nucleotides,preferably about 15-35 nucleotides, more preferably 18-28 nucleotides,which are identical or complementary to and therefor hybridize to theGCC gene transcript or cDNA generated therefrom. In preferredembodiments, the primers are each 15-35 nucleotide, more preferably18-28 nucleotide fragments of SEQ ID NO:1. The primer must hybridize tothe sequence to be amplified. Typical primers are 18-28 nucleotides inlength and are generally have 50% to 60% G+C composition. The entireprimer is preferably complementary to the sequence it must hybridize to.Preferably, primers generate PCR products 100 base pairs to 2000 basepairs. However, it is possible to generate products of 50 to up to 10 kband more. If mRNA is used as a template, the primers must hybridize tomRNA sequences. If cDNA is used as a template, the primers musthybridize to cDNA sequences.

The mRNA or cDNA is combined with the primers, free nucleotides andenzyme following standard PCR protocols. The mixture undergoes a seriesof temperature changes. If the GCC gene transcript or cDNA generatedtherefrom is present, that is, if both primers hybridize to sequences onthe same molecule, the molecule comprising the primers and theintervening complementary sequences will be exponentially amplified.

The amplified DNA can be easily detected by a variety of well knownmeans. If no GCC gene transcript or cDNA generated therefrom is present,no PCR product will be exponentially amplified. The PCR technologytherefore provides an extremely easy, straightforward and reliablemethod of detecting the GCC gene transcript in a sample.

PCR product may be detected by several well known means. The preferredmethod for detecting the presence of amplified DNA is to separate thePCR reaction material by gel electrophoresis and stain the gel withethidium bromide in order to visual the amplified DNA if present. A sizestandard of the expected size of the amplified DNA is preferably run onthe gel as a control.

In some instances, such as when unusually small amounts of RNA arerecovered and only small amounts of cDNA are generated therefrom, it isdesirable or necessary to perform a PCR reaction on the first PCRreaction product. That is, if difficult to detect quantities ofamplified DNA are produced by the first reaction, a second PCR can beperformed to make multiple copies of DNA sequences of the firstamplified DNA. A nested set of primers are used in the second PCRreaction. The nested set of primers hybridize to sequences downstream ofthe 5′ primer and upstream of the 3′ primer used in the first reaction.

The present invention includes oligonucleotide which are useful asprimers for performing PCR methods to amplify the GCC gene transcript orcDNA generated therefrom.

According to the invention, diagnostic kits can be assembled which areuseful to practice methods of detecting the presence of the GCC genetranscript or cDNA generated therefrom in non-colorectal samples. Suchdiagnostic kits comprise oligonucleotide which are useful as primers forperforming PCR methods. It is preferred that diagnostic kits accordingto the present invention comprise a container comprising a size markerto be run as a standard on a gel used to detect the presence ofamplified DNA. The size marker is the same size as the DNA generated bythe primers in the presence of the GCC gene transcript or cDNA generatedtherefrom. Additional components in some kits include instructions forcarrying out the assay. Additionally the kit may optionally comprisedepictions or photographs that represent the appearance of positive andnegative results. Positive and negative controls may also be provided.

PCR assays are useful for detecting the GCC gene transcript inhomogenized tissue samples and cells in body fluid samples. It iscontemplated that PCR on the plasma portion of a fluid sample could beused to detect the GCC gene transcript.

Another method of determining whether a sample contains cells expressingGCC is by branched chain oligonucleotide hybridization analysis of mRNAextracted from a sample. Branched chain oligonucleotide hybridizationmay be performed as described in U.S. Pat. No. 5,597,909, U.S. Pat. No.5,437,977 and U.S. Pat. No. 5,430,138, which are each incorporatedherein by reference. Reagents may be designed following the teachings ofthose patents and that sequence of the GCC gene transcript.

Another method of determining whether a sample contains cells expressingGCC is by Northern Blot analysis of mRNA extracted from a non-colorectalsample. The techniques for performing Northern blot analyses are wellknown by those having ordinary skill in the art and are described inSambrook, J. et al., (1989) Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. mRNAextraction, electrophoretic separation of the mRNA, blotting, probepreparation and hybridization are all well known techniques that can beroutinely performed using readily available starting material.

The mRNA is extracted using poly dT columns and the material isseparated by electrophoresis and, for example, transferred tonitrocellulose paper. Labeled probes made from an isolated specificfragment or fragments can be used to visualize the presence of acomplementary fragment fixed to the paper. Probes useful to identifymRNA in a Northern Blot have a nucleotide sequence that is complementaryto the GCC gene transcript. Those having ordinary skill in the art coulduse the sequence information in SEQ ID NO: 1 to design such probes or toisolate and clone the GCC gene transcript or cDNA generated therefrom tobe used as a probe. Such probes are at least 15 nucleotides, preferably30-200, more preferably 40-100 nucleotide fragments and may be theentire GCC gene transcript.

According to the invention, diagnostic kits can be assembled which areuseful to practice methods of detecting the presence of the GCC genetranscript in non-colorectal samples by Northern blot analysis. Suchdiagnostic kits comprise oligonucleotide which are useful as probes forhybridizing to the mRNA. The probes may be radiolabeled. It is preferredthat diagnostic kits according to the present invention comprise acontainer comprising a size marker to be run as a standard on a gel. Itis preferred that diagnostic kits according to the present inventioncomprise a container comprising a positive control which will hybridizeto the probe. Additional components in some kits include instructionsfor carrying out the assay. Additionally the kit may optionally comprisedepictions or photographs that represent the appearance of positive andnegative results.

Northern blot analysis is useful for detecting the GCC gene transcriptin homogenized tissue samples and cells in body fluid samples. It iscontemplated that PCR on the plasma portion of a fluid sample could beused to detect the GCC gene transcript.

Another method of detecting the presence of the GCC gene transcript byoligonucleotide hybridization technology. Oligonucleotide hybridizationtechnology is well known to those having ordinary skill in the art.Briefly, detectable probes which contain a specific nucleotide sequencethat will hybridize to nucleotide sequence of the GCC gene transcript.RNA or cDNA made from RNA from a sample is fixed, usually to filterpaper or the like. The probes are added and maintained under conditionsthat permit hybridization only if the probes fully complement the fixedgenetic material. The conditions are sufficiently stringent to wash offprobes in which only a portion of the probe hybridizes to the fixedmaterial. Detection of the probe on the washed filter indicatecomplementary sequences.

Probes useful in oligonucleotide assays at least 18 nucleotides ofcomplementary DNA and may be as large as a complete complementarysequence to the GCC gene transcript. In some preferred embodiments theprobes of the invention are 30-200 nucleotides, preferably 40-100nucleotides.

One having ordinary skill in the art, using the sequence informationdisclosed in SEQ ID NO:1 can design probes useful in the invention.Hybridization conditions can be routinely optimized to minimizebackground signal by non-fully complementary hybridization. In somepreferred embodiments, the probes are full length clones. Probes are atleast 15 nucleotides, preferably 30-200, more preferably 40-100nucleotide fragments and may be the entire GCC gene transcript.

The present invention includes labeled oligonucleotide which are usefulas probes for performing oligonucleotide hybridization. The labeledprobes of the present invention are labeled with radiolabelednucleotides or are otherwise detectable by readily availablenonradioactive detection systems.

According to the invention, diagnostic kits can be assembled which areuseful to practice oligonucleotide hybridization methods of theinvention. Such diagnostic kits comprise a labeled oligonucleotide whichencodes portions of the GCC gene transcript.

It is preferred that labeled probes of the oligonucleotide diagnostickits according to the present invention are labeled with aradionucleotide. The oligonucleotide hybridization-based diagnostic kitsaccording to the invention preferably comprise DNA samples thatrepresent positive and negative controls. A positive control DNA sampleis one that comprises a nucleic acid molecule which has a nucleotidesequence that is fully complementary to the probes of the kit such thatthe probes will hybridize to the molecule under assay conditions. Anegative control DNA sample is one that comprises at least one nucleicacid molecule, the nucleotide sequence of which is partiallycomplementary to the sequences of the probe of the kit. Under assayconditions, the probe will not hybridize to the negative control DNAsample. Additional components in some kits include instructions forcarrying out the assay. Additionally the kit may optionally comprisedepictions or photographs that represent the appearance of positive andnegative results.

Oligonucleotide hybridization techniques are useful for detecting theGCC gene transcript in homogenized tissue samples and cells in bodyfluid samples. It is contemplated that PCR on the plasma portion of afluid sample could be used to detect the GCC gene transcript.

The present invention relates to in vitro kits for evaluating samples oftumors to determine whether or not they are stomach or esophageal inorigin and to reagents and compositions useful to practice the same. Insome embodiments of the invention, tumor samples may be isolated fromindividuals undergoing or recovery from surgery to remove tumors in thestomach or esophagus, tumors in other organs or biopsy material. Thetumor sample is analyzed to identify the presence or absence of the GCCgene transcript.

Techniques such as immunohistochemistry assays may be performed todetermine whether GCC are present in cells in the tumor sample. Thepresence of mRNA that encodes the GCC protein or cDNA generatedtherefrom can be determined using techniques such as in situhybridization, immunohistochemistry and in situ ST binding assay.

In situ hybridization technology is well known by those having ordinaryskill in the art. Briefly, cells are fixed and detectable probes whichcontain a specific nucleotide sequence are added to the fixed cells. Ifthe cells contain complementary nucleotide sequences, the probes, whichcan be detected, will hybridize to them.

Probes useful in oligonucleotide assays at least 18 nucleotides ofcomplementary DNA and may be as large as a complete complementarysequence to the GCC gene transcript. In some preferred embodiments theprobes of the invention are 30-200 nucleotides, preferably 40-100nucleotides.

One having ordinary skill in the art, using the sequence information setforth in SEQ ID NO: 1 can design probes useful in in situ hybridizationtechnology to identify cells that express GCC. Probes preferablyhybridizes to a nucleotide sequence that corresponds to the GCC genetranscript. Hybridization conditions can be routinely optimized tominimize background signal by non-fully complementary hybridization.

Probes preferably hybridize to the full length GCC gene transcript.Probes are at least 15 nucleotides, preferably 30-200, more preferably40-100 nucleotide fragments and may be the GCC gene transcript, morepreferably 18-28 nucleotide fragments of the GCC gene transcript.

The probes are fully complementary and do not hybridize well topartially complementary sequences. For in situ hybridization accordingto the invention, it is preferred that the probes are detectable byfluorescence. A common procedure is to label probe with biotin-modifiednucleotide and then detect with fluorescently tagged avidin.

Hence, probe does not itself have to be labeled with florescent but canbe subsequently detected with florescent marker.

The present invention includes labeled oligonucleotide which are usefulas probes for performing oligonucleotide hybridization. That is, theyare fully complementary with mRNA sequences but not genomic sequences.The labeled probes of the present invention are labeled withradiolabeled nucleotides or are otherwise detectable by readilyavailable nonradioactive detection systems.

The present invention relates to probes useful for in situ hybridizationto identify cells that express GCC.

Cells are fixed and the probes are added to the genetic material. Probeswill hybridize to the complementary nucleic acid sequences present inthe sample. Using a fluorescent microscope, the probes can be visualizedby their fluorescent markers.

According to the invention, diagnostic kits can be assembled which areuseful to practice in situ hybridization methods of the invention arefully complementary with mRNA sequences but not genomic sequences. Forexample, the mRNA sequence includes different exon sequences. It ispreferred that labeled probes of the in situ diagnostic kits accordingto the present invention are labeled with a fluorescent marker.

Immunohistochemistry techniques may be used to identify and essentiallystain cells with GCC. Such “staining” allows for analysis of metastaticmigration. Anti-GCC antibodies such as those described above ofcontacted with fixed cells and the GCC present in the cells reacts withthe antibodies. The antibodies are detectably labeled or detected usinglabeled second antibody or protein A to stain the cells.

The techniques described herein for evaluating tumor sections can alsobe used to analyze tissue sections for samples of lymph nodes as well asother tissues to identify the presence of cells that express GCC. Thesamples can be prepared and “stained” to detect expression of GCC.

Immunoassay methods may be used in the diagnosis of individualssuffering from stomach or esophageal cancer by detecting presence of GCCin sample of non-colorectal tissue or body fluid from an individualssuspected of having or being susceptible to stomach or esophageal cancerusing antibodies which were produced in response to exposure to such GCCprotein. Moreover, immunoassay methods may be used to identifyindividuals suffering from stomach or esophageal cancer by detectingpresence of GCC in sample of tumor using antibodies which were producedin response to exposure to such GCC protein.

The antibodies are preferably monoclonal antibodies. The antibodies arepreferably raised against GCC made in human cells. Immunoassays are wellknown and there design may be routinely undertaken by those havingordinary skill in the art. Those having ordinary skill in the art canproduce monoclonal antibodies which specifically bind to GCC and areuseful in methods and kits of the invention using standard techniquesand readily available starting materials. The techniques for producingmonoclonal antibodies are outlined in Harlow, E. and D. Lane, (1988)ANTIBODIES: A Laboratory Manual, Cold Spring Harbor Laboratory, ColdSpring Harbor N.Y., which is incorporated herein by reference, providedetailed guidance for the production of hybridomas and monoclonalantibodies which specifically bind to target proteins. It is within thescope of the present invention to include Fabs, recombinant Fabs,F(Ab)2s, recombinant F(Ab)2s which specifically bind to GCC translationproducts in place of antibodies.

Briefly, GCC protein is injected into mice. The spleen of the mouse isremoved, the spleen cells are isolated and fused with immortalized mousecells. The hybrid cells, or hybridomas, are cultured and those cellswhich secrete antibodies are selected. The antibodies are analyzed and,if found to specifically bind to the GCC, the hybridoma which producesthem is cultured to produce a continuous supply of anti-GCC specificantibodies.

The antibodies are preferably monoclonal antibodies. The antibodies arepreferably raised against GCC made in human cells.

The means to detect the presence of a protein in a test sample areroutine and one having ordinary skill in the art can detect the presenceor absence of a protein or an antibody using well known methods. Onewell known method of detecting the presence of a protein is animmunoassay. One having ordinary skill in the art can readily appreciatethe multitude of ways to practice an immunoassay to detect the presenceof a GCC protein in a sample.

According to some embodiments, immunoassays comprise allowing proteinsin the sample to bind a solid phase support such as a plastic surface.Detectable antibodies are then added which selectively binding to theGCC. Detection of the detectable antibody indicates the presence of GCC.The detectable antibody may be a labeled or an unlabeled antibody.Unlabeled antibody may be detected using a second, labeled antibody thatspecifically binds to the first antibody or a second, unlabeled antibodywhich can be detected using labeled protein A, a protein that complexeswith antibodies. Various immunoassay procedures are described inImmunoassays for the 80's, A. Voller et al., Eds., University Park,1981, which is incorporated herein by reference.

Simple immunoassays may be performed in which a solid phase support iscontacted with the test sample. Any proteins present in the test samplebind the solid phase support and can be detected by a specific,detectable antibody preparation. Such a technique is the essence of thedot blot, Western blot and other such similar assays.

Other immunoassays may be more complicated but actually provideexcellent results. Typical and preferred immunometric assays include“forward” assays for the detection of a protein in which a firstanti-protein antibody bound to a solid phase support is contacted withthe test sample. After a suitable incubation period, the solid phasesupport is washed to remove unbound protein. A second, distinctanti-protein antibody is then added which is specific for a portion ofthe specific protein not recognized by the first antibody. The secondantibody is preferably detectable. After a second incubation period topermit the detectable antibody to complex with the specific proteinbound to the solid phase support through the first antibody, the solidphase support is washed a second time to remove the unbound detectableantibody. Alternatively, the second antibody may not be detectable. Inthis case, a third detectable antibody, which binds the second antibodyis added to the system. This type of “forward sandwich” assay may be asimple yes/no assay to determine whether binding has occurred or may bemade quantitative by comparing the amount of detectable antibody withthat obtained in a control. Such “two-site” or “sandwich” assays aredescribed by Wide, Radioimmune Assay Method, Kirkham, Ed., E. & S.Livingstone, Edinburgh, 1970, pp. 199-206, which is incorporated hereinby reference.

Other types of immunometric assays are the so-called “simultaneous” and“reverse” assays. A simultaneous assay involves a single incubation stepwherein the first antibody bound to the solid phase support, the second,detectable antibody and the test sample are added at the same time.After the incubation is completed, the solid phase support is washed toremove unbound proteins. The presence of detectable antibody associatedwith the solid support is then determined as it would be in aconventional “forward sandwich” assay. The simultaneous assay may alsobe adapted in a similar manner for the detection of antibodies in a testsample.

The “reverse” assay comprises the stepwise addition of a solution ofdetectable antibody to the test sample followed by an incubation periodand the addition of antibody bound to a solid phase support after anadditional incubation period. The solid phase support is washed inconventional fashion to remove unbound protein/antibody complexes andunreacted detectable antibody. The determination of detectable antibodyassociated with the solid phase support is then determined as in the“simultaneous” and “forward” assays. The reverse assay may also beadapted in a similar manner for the detection of antibodies in a testsample.

The first component of the immunometric assay may be added tonitrocellulose or other solid phase support which is capable ofimmobilizing proteins. The first component for determining the presenceof GCC in a test sample is an anti-GCC antibody. By “solid phasesupport” or “support” is intended any material capable of bindingproteins. Well-known solid phase supports include glass, polystyrene,polypropylene, polyethylene, dextran, nylon, amylases, natural andmodified celluloses, polyacrylamides, agaroses, and magnetite. Thenature of the support can be either soluble to some extent or insolublefor the purposes of the present invention. The support configuration maybe spherical, as in a bead, or cylindrical, as in the inside surface ofa test tube or the external surface of a rod. Alternatively, the surfacemay be flat such as a sheet, test strip, etc. Those skilled in the artwill know many other suitable “solid phase supports” for bindingproteins or will be able to ascertain the same by use of routineexperimentation.

A preferred solid phase support is a 96-well microtiter plate.

To detect the presence of GCC, detectable anti-GCC antibodies are used.

Several methods are well known for the detection of antibodies.

One method in which the antibodies can be detectably labeled is bylinking the antibodies to an enzyme and subsequently using theantibodies in an enzyme immunoassay (EIA) or enzyme-linked immunosorbentassay (ELISA), such as a capture ELISA. The enzyme, when subsequentlyexposed to its substrate, reacts with the substrate and generates achemical moiety which can be detected, for example, byspectrophotometric, fluorometric or visual means. Enzymes which can beused to detectably label antibodies include, but are not limited tomalate dehydrogenase, staphylococcal nuclease, delta-5-steroidisomerase, yeast alcohol dehydrogenase, alpha-glycerophosphatedehydrogenase, triose phosphate isomerase, horseradish peroxidase,alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase,ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,glucoamylase and acetylcholinesterase. One skilled in the art wouldreadily recognize other enzymes which may also be used.

Another method in which antibodies can be detectably labeled is throughradioactive isotopes and subsequent use in a radioimmunoassay (RIA)(see, for example, Work, T. S. et al., Laboratory Techniques andBiochemistry in Molecular Biology, North Holland Publishing Company,N.Y., 1978, which is incorporated herein by reference). The radioactiveisotope can be detected by such means as the use of a gamma counter or ascintillation counter or by autoradiography. Isotopes which areparticularly useful for the purpose of the present invention are ³H,¹²⁵I, ¹³¹I, ³⁵S, and ¹⁴C. Preferably ¹²⁵I is the isotope. One skilled inthe art would readily recognize other radioisotopes which may also beused.

It is also possible to label the antibody with a fluorescent compound.When the fluorescent-labeled antibody is exposed to light of the properwave length, its presence can be detected due to its fluorescence. Amongthe most commonly used fluorescent labeling compounds are fluoresceinisothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin,o-phthaldehyde and fluorescamine. One skilled in the art would readilyrecognize other fluorescent compounds which may also be used.

Antibodies can also be detectably labeled using fluorescence-emittingmetals such as ¹⁵²Eu, or others of the lanthanide series. These metalscan be attached to the protein-specific antibody using such metalchelating groups as diethylenetriaminepentaacetic acid (DTPA) orethylenediamine-tetraacetic acid (EDTA). One skilled in the art wouldreadily recognize other fluorescence-emitting metals as well as othermetal chelating groups which may also be used.

Antibody can also be detectably labeled by coupling to achemiluminescent compound. The presence of the chemiluminescent-labeledantibody is determined by detecting the presence of luminescence thatarises during the course of a chemical reaction. Examples ofparticularly useful chemoluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester. One skilled in the art would readily recognize otherchemiluminescent compounds which may also be used.

Likewise, a bioluminescent compound may be used to label antibodies.

Bioluminescence is a type of chemiluminescence found in biologicalsystems in which a catalytic protein increases the efficiency of thechemiluminescent reaction. The presence of a bioluminescent protein isdetermined by detecting the presence of luminescence. Importantbioluminescent compounds for purposes of labeling are luciferin,luciferase and aequorin. One skilled in the art would readily recognizeother bioluminescent compounds which may also be used.

Detection of the protein-specific antibody, fragment or derivative maybe accomplished by a scintillation counter if, for example, thedetectable label is a radioactive gamma emitter. Alternatively,detection may be accomplished by a fluorometer if, for example, thelabel is a fluorescent material. In the case of an enzyme label, thedetection can be accomplished by colorometric methods which employ asubstrate for the enzyme.

Detection may also be accomplished by visual comparison of the extent ofenzymatic reaction of a substrate in comparison with similarly preparedstandards. One skilled in the art would readily recognize otherappropriate methods of detection which may also be used.

The binding activity of a given lot of antibodies may be determinedaccording to well known methods. Those skilled in the art will be ableto determine operative and optimal assay conditions for eachdetermination by employing routine experimentation.

Positive and negative controls may be performed in which known amountsof GCC proteins and no GCC protein, respectively, are added to assaysbeing performed in parallel with the test assay. One skilled in the artwould have the necessary knowledge to perform the appropriate controls.In addition, the kit may comprise instructions for performing the assay.Additionally the kit may optionally comprise depictions or photographsthat represent the appearance of positive and negative results.

GCC may be produced as a reagent for positive controls routinely. Oneskilled in the art would appreciate the different manners in which theGCC protein may be produced and isolated.

Antibody composition refers to the antibody or antibodies required forthe detection of the protein. For example, the antibody composition usedfor the detection of a GCC in a test sample comprises a first antibodythat binds to the GCC as well as a second or third detectable antibodythat binds the first or second antibody, respectively.

To examine a test sample for the presence of a GCC, a standardimmunometric assay such as the one described below may be performed. Afirst anti-GCC antibody, which recognizes a specific portion of GCC, isadded to a 96-well microtiter plate in a volume of buffer. The plate isincubated for a period of time sufficient for binding to occur andsubsequently washed with PBS to remove unbound antibody. The plate isthen blocked with a PBS/BSA solution to prevent sample proteins fromnon-specifically binding the microtiter plate. Test sample aresubsequently added to the wells and the plate is incubated for a periodof time sufficient for binding to occur. The wells are washed with PBSto remove unbound protein. Labeled anti-GCC antibodies, which recognizeportions of GCC not recognized by the first antibody, are added to thewells. The plate is incubated for a period of time sufficient forbinding to occur and subsequently washed with PBS to remove unbound,labeled anti-GCC antibody. The amount of labeled and bound anti-GCCantibody is subsequently determined by standard techniques.

Kits which are useful for the detection of GCC in a test sample comprisea container comprising anti-GCC antibodies and a container or containerscomprising controls. Controls include one control sample which does notcontain GCC and/or another control sample which contained the GCC. Theanti-GCC antibodies used in the kit are detectable such as beingdetectably labeled. If the detectable anti-GCC antibody is not labeled,it may be detected by second antibodies or protein A for example whichmay also be provided in some kits in separate containers. Additionalcomponents in some kits include solid support, buffer, and instructionsfor carrying out the assay. Additionally the kit may optionally comprisedepictions or photographs that represent the appearance of positive andnegative results.

The immunoassay is useful for detecting GCC in homogenized tissuesamples and body fluid samples including the plasma portion or cells inthe fluid sample.

Western Blots may be useful in assisting the diagnosis os individualssuffering from stomach or esophageal cancer by detecting presence of GCCof non-colorectal tissue or body fluid. Western blots may also be usedto detect presence of GCC in sample of tumor from an individualsuffering from cancer. Western blots use detectable anti-GCC—antibodiesto bind to any GCC present in a sample and thus indicate the presence ofthe receptor in the sample.

Western blot techniques, which are described in Sambrook, J. et al.,(1989) Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., which is incorporated hereinby reference, are similar to immunoassays with the essential differencebeing that prior to exposing the sample to the antibodies, the proteinsin the samples are separated by gel electrophoresis and the separatedproteins are then probed with antibodies. In some preferred embodiments,the matrix is an SDS-PAGE gel matrix and the separated proteins in thematrix are transferred to a carrier such as filter paper prior toprobing with antibodies. Anti-GCC antibodies described above are usefulin Western blot methods.

Generally, samples are homogenized and cells are lysed using detergentsuch as Triton-X. The material is then separated by the standardtechniques in Sambrook, J. et al., (1989) Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.

Kits which are useful for the detection of GCC in a test sample byWestern Blot comprise a container comprising anti-GCC antibodies and acontainer or containers comprising controls. Controls include onecontrol sample which does not contain GCC and/or another control samplewhich contains GCC. The anti-GCC antibodies used in the kit aredetectable such as being detectably labeled. If the detectable anti-GCCantibody is not labeled, it may be detected by second antibodies orprotein A for example which may also be provided in some kits inseparate containers. Additional components in some kits includeinstructions for carrying out the assay. Additionally the kit mayoptionally comprise depictions or photographs that represent theappearance of positive and negative results.

Western blots are useful for detecting GCC in homogenized tissue samplesand body fluid samples including the plasma portion or cells in thefluid sample.

In Vivo Imaging and Therapeutics

According to some embodiments of the invention, compositions and in vivomethods are provided for detecting, imaging, or treating primary and/ormetastatic stomach or esophageal tumors in an individual.

When the conjugated compositions of the present invention areadministered outside the intestinal tract such as when administered inthe circulatory system, they remain segregated from the cells that linethe intestinal tract and will bind only to cells outside the intestinaltract which express GCC. The conjugated compositions will not bind tothe normal cells but will bind to primary and/or metastatic stomach oresophageal cells.

Thus, the active moieties of conjugated compositions administeredoutside the intestinal tract are delivered to cells which express GCCsuch as primary and/or metastatic stomach or esophageal cancer cells.

Therapeutic and diagnostic pharmaceutical compositions useful in thepresent invention include conjugated compounds that specifically targetcells that express GCC.

These conjugated compounds include moieties that bind to GCC which donot bind to cells of normal tissue in the body except cells of theintestinal tract since the cells of other tissues do not express GCC.

Unlike normal colorectal cells, cancer cells that express GCC areaccessible to substances administered outside the intestinal tract, forexample administered in the circulatory system. The only GCC in normaltissue exist in the apical membranes of intestinal mucosa cells and thuseffectively isolated from the targeted cancer chemotherapeutics andimaging agents administered outside the intestinal tract by theintestinal mucosa barrier. Thus, primary and/or metastatic stomach oresophageal cancer cells may be targeted by conjugated compounds of thepresent invention by introducing such compounds outside the intestinaltract such as for example by administering pharmaceutical compositionsthat comprise conjugated compounds into the circulatory system.

One having ordinary skill in the art can identify individuals suspectedof suffering from primary and/or metastatic stomach or esophagealcancer. In those individuals diagnosed with stomach or esophagealcancer, it is not unusual and in some cases standard therapy to suspectmetastasis and aggressively attempt to eradicate metastasized cells. Thepresent invention provides pharmaceutical compositions and methods forimaging and thereby will more definitively diagnose primary andmetastastic disease. Further, the present invention providespharmaceutical compositions comprising therapeutic agents and methodsfor specifically targeting and eliminating primary and/or metastaticstomach or esophageal cancer cells. Further, the present inventionprovides pharmaceutical compositions that comprise therapeutics andmethods for specifically eliminating primary and/or metastatic stomachor esophageal cancer cells.

The pharmaceutical compositions which comprise conjugated compositionsof the present invention may be used to diagnose or treat individualssuffering from primary and/or metastatic stomach or esophageal tumors.

The present invention relies upon the use of a GCC binding moiety in aconjugated composition. The GCC binding moiety is essentially a portionof the conjugated composition which acts as a ligand to a GCC and thusspecifically binds to it.

The conjugated composition also includes an active moiety which isassociated with the GCC binding moiety; the active moiety being anactive agent which is either useful to image, target, neutralize or killthe cell.

According to the present invention, the GCC binding moiety is the GCCligand portion of a conjugated composition. In some embodiments, the GCCligand is an antibody.

In some preferred embodiments, conjugated compounds comprise GCC bindingmoieties that comprise an anti-GCC antibody.

It is preferred that the GCC ligand used as the GCC binding moiety be assmall as possible. Thus it is preferred that the GCC ligand be anon-peptide small molecule or small peptide, preferably less than 25amino acids, more preferably less than 20 amino acids. In someembodiments, the GCC ligand which constitute the GCC binding moiety of aconjugated composition is less than 15 amino acids. GCC binding peptidecomprising less than 10 amino acids and GCC binding peptide less than 5amino acids may be used as GCC binding moieties according to the presentinvention. It is within the scope of the present invention to includelarger molecules which serve as GCC binding moieties including, but notlimited to molecules such as antibodies which specifically bind to GCC.

GCC ligands useful as GCC binding moieties may be identified usingvarious well known combinatorial library screening technologies such asthose set forth in Example 1 herein.

An assay may be used to test both peptide and non-peptide compositionsto determine whether or not they are GCC ligands or, to test conjugatedcompositions to determine if they possess GCC—binding activity. Suchcompositions that specifically bind to GCC can be identified by acompetitive binding assay using antibodies known to bind to the GCC. Thecompetitive binding assay is a standard technique in pharmacology whichcan be readily performed by those having ordinary skill in the art usingreadily available starting materials.

GCC may be produced synthetically, recombinantly or isolated fromnatural sources.

Using a solid phase synthesis as an example, the protected orderivatized amino acid is attached to an inert solid support through itsunprotected carboxyl or amino group. The protecting group of the aminoor carboxyl group is then selectively removed and the next amino acid inthe sequence having the complementary (amino or carboxyl) group suitablyprotected is admixed and reacted with the residue already attached tothe solid support. The protecting group of the amino or carboxyl groupis then removed from this newly added amino acid residue, and the nextamino acid (suitably protected) is then added, and so forth. After allthe desired amino acids have been linked in the proper sequence, anyremaining terminal and side group protecting groups (and solid support)are removed sequentially or concurrently, to provide the final peptide.The peptide of the invention are preferably devoid of benzylated ormethylbenzylated amino acids. Such protecting group moieties may be usedin the course of synthesis, but they are removed before the peptides areused. Additional reactions may be necessary, as described elsewhere, toform intramolecular linkages to restrain conformation.

Antibodies against GCC may be routinely produced and used in competitionassays to identify GCC ligands or as starting materials for conjugatedcompounds according to the invention.

According to the present invention, the active moiety may be atherapeutic agent or an imaging agent. One having ordinary skill in theart can readily recognize the advantages of being able to specificallytarget cancer cells with an GCC ligand and conjugate such a ligand withmany different active agents.

Chemotherapeutics useful as active moieties which when conjugated to aGCC binding moiety are specifically delivered to cells that express GCCsuch as stomach or esophageal cancer cells, are typically small chemicalentities produced by chemical synthesis. Chemotherapeutics includecytotoxic and cytostatic drugs. Chemotherapeutics may include thosewhich have other effects on cells such as reversal of the transformedstate to a differentiated state or those which inhibit cell replication.Examples of chemotherapeutics include common cytotoxic or cytostaticdrugs such as for example: methotrexate (amethopterin), doxorubicin(adrimycin), daunorubicin, cytosinarabinoside, etoposide, 5-4fluorouracil, melphalan, chlorambucil, and other nitrogen mustards (e.g.cyclophosphamide), cis-platinum, vindesine (and other vinca alkaloids),mitomycin and bleomycin. Other chemotherapeutics include: purothionin(barley flour oligopeptide), macromomycin. 1,4-benzoquinone derivativesand trenimon.

Toxins are useful as active moieties. When a toxin is conjugated to aGCC binding moiety, the conjugated composition is specifically deliveredto a cell that expresses GCC such as stomach or esophageal cancer cellsby way of the GCC binding moiety and the toxin moiety kills the cell.Toxins are generally complex toxic products of various organismsincluding bacteria, plants, etc. Examples of toxins include but are notlimited to: ricin, ricin A chain (ricin toxin), Pseudomonas exotoxin(PE), diphtheria toxin (DT), Clostridium perfringens phospholipase C(PLC), bovine pancreatic ribonuclease (BPR), pokeweed antiviral protein(PAP), abrin, abrin A chain (abrin toxin), cobra venom factor (CVF),gelonin (GEL), saporin (SAP), modeccin, viscumin and volkensin. Asdiscussed above, when protein toxins are employed with GCC bindingpeptides, conjugated compositions may be produced using recombinant DNAtechniques. Briefly, a recombinant DNA molecule can be constructed whichencodes both the GCC ligand and the toxin on a chimeric gene. When thechimeric gene is expressed, a fusion protein is produced which includesa GCC binding moiety and an active moiety. Protein toxins are alsouseful to form conjugated compounds with GCC binding peptides throughnon-peptidyl bonds.

In addition, there are other approaches for utilizing active agents forthe treatment of cancer. For example, conjugated compositions may beproduced which include a GCC binding moiety and an active moiety whichis an active enzyme. The GCC binding moiety specifically localizes theconjugated composition to the tumor cells. An inactive prodrug which canbe converted by the enzyme into an active drug is administered to thepatient. The prodrug is only converted to an active drug by the enzymewhich is localized to the tumor. An example of an enzyme/prodrug pairincludes alkaline phosphatase/etoposidephosphate. In such a case, thealkaline phosphatase is conjugated to a GCC binding ligand. Theconjugated compound is administered and localizes at the cancer cell.Upon contact with etoposidephosphate (the prodrug), theetoposidephosphate is converted to etoposide, a chemotherapeutic drugwhich is taken up by the cancer cell.

Radiosensitizing agents are substances that increase the sensitivity ofcells to radiation. Examples of radiosensitizing agents includenitroimidazoles, metronidazole and misonidazole (see: DeVita, V. T. Jr.in Harrison's Principles of Internal Medicine, p. 68, McGraw-Hill BookCo., N.Y. 1983, which is incorporated herein by reference). Theconjugated compound that comprises a radiosensitizing agent as theactive moiety is administered and localizes at the primary and/ormetastatic stomach or esophageal cancer cell. Upon exposure of theindividual to radiation, the radiosensitizing agent is “excited” andcauses the death of the cell.

Radionuclides may be used in pharmaceutical compositions that are usefulfor radiotherapy or imaging procedures.

Examples of radionuclides useful as toxins in radiation therapy include:⁴⁷Sc, ⁶⁷Cu, ⁹⁰Y, ¹⁰⁹Pd, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁹⁹Au, ²¹¹At,²¹²Pb and ²¹²B. Other radionuclides which have been used by those havingordinary skill in the art include: ³²P and ³³P, ⁷¹Ge, ⁷⁷As, ¹⁰³Pb,¹⁰⁵Rh, ¹¹¹Ag, ¹¹⁹Sb, ¹²¹Sn, ¹³¹Cs, ¹⁴³Pr, ¹⁶¹Tb, ¹⁷⁷Lu, ¹⁹¹Os,^(193M)Pt, ¹⁹⁷Hg, all beta negative and/or auger emitters. Somepreferred radionuclides include: ⁹⁰Y, ¹³¹I²¹¹At and ²¹²Pb/²¹²Bi.

According to the present invention, the active moieties may be animaging agent. Imaging agents are useful diagnostic procedures as wellas the procedures used to identify the location of cancer cells. Imagingcan be performed by many procedures well-known to those having ordinaryskill in the art and the appropriate imaging agent useful in suchprocedures may be conjugated to a GCC ligand by well-known means.Imaging can be performed, for example, by radioscintigraphy, nuclearmagnetic resonance imaging (MRI) or computed tomography (CT scan). Themost commonly employed radionuclide imaging agents include radioactiveiodine and indium. Imaging by CT scan may employ a heavy metal such asiron chelates. MRI scanning may employ chelates of gadolinium ormanganese. Additionally, positron emission tomography (PET) may bepossible using positron emitters of oxygen, nitrogen, iron, carbon, orgallium. Example of radionuclides useful in imaging procedures include:⁴³K, ⁵²Fe, ⁵⁷Co, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁷Br, ⁸¹Rb/^(81M)Kr, ^(87M)Sr,^(99M)Tc, ¹¹¹In, ^(113M)In, ¹²³I, ¹²⁵I, ¹²⁷Cs, ¹²⁹Cs, ¹³¹I, ¹³²I, ¹⁹⁷Hg,²⁰³Pb and ²⁰⁶Bi.

It is preferred that the conjugated compositions be non-immunogenic orimmunogenic at a very low level. Accordingly, it is preferred that theGCC binding moiety be a small, poorly immunogenic or non-immunogenicpeptide or a non-peptide. The GCC binding moiety may be a humanized orprimatized antibody or a human antibody.

GCC ligands are conjugated to active agents by a variety of well-knowntechniques readily performed without undue experimentation by thosehaving ordinary skill in the art. The technique used to conjugate theGCC ligand to the active agent is dependent upon the molecular nature ofthe GCC ligand and the active agent. After the GCC ligand and the activeagent are conjugated to form a single molecule, assays may be performedto ensure that the conjugated molecule retains the activities of themoieties. The competitive binding assay described above may be used toconfirm that the GCC binding moiety retains its binding activity as aconjugated compound. Similarly, the activity of the active moiety may betested using various assays for each respective type of active agent.Radionuclides retain there activity, i.e. their radioactivity,irrespective of conjugation. With respect to active agents which aretoxins, drugs and targeting agents, standard assays to demonstrate theactivity of unconjugated forms of these compounds may be used to confirmthat the activity has been retained.

Conjugation may be accomplished directly between the GCC ligand and theactive agent or linking, intermediate molecular groups may be providedbetween the GCC ligand and the active agent. Crosslinkers areparticularly useful to facilitate conjugation by providing attachmentsites for each moiety. Crosslinkers may include additional moleculargroups which serve as spacers to separate the moieties from each otherto prevent either from interfering with the activity of the other.

One having ordinary skill in the art may conjugate a GCC ligand to achemotherapeutic drug using well-known techniques. For example,Magerstadt, M. Antibody Conjugates and Malignant Disease. (1991) CRCPress, Boca Raton, USA, pp. 110-152) which is incorporated herein byreference, teaches the conjugation of various cytostatic drugs to aminoacids of antibodies. Such reactions may be applied to conjugatechemotherapeutic drugs to GCC ligands, including anti-GCC antibodies,with an appropriate linker. Most of the chemotherapeutic agentscurrently in use in treating cancer possess functional groups that areamenable to chemical crosslinking directly with proteins. For example,free amino groups are available on methotrexate, doxorubicin,daunorubicin, cytosinarabinoside, cis-platin, vindesine, mitomycin andbleomycin while free carboxylic acid groups are available onmethotrexate, melphalan, and chlorambucil.

These functional groups, that is free amino and carboxylic acids, aretargets for a variety of homobifunctional and heterobifunctionalchemical crosslinking agents which can crosslink these drugs directly tothe single free amino group of an antibody. For example, one procedurefor crosslinking GCC ligands which have a free amino group to activeagents which have a free amino group such as methotrexate, doxorubicin,daunorubicin, cytosinarabinoside, cis-platin, vindesine, mitomycin andbleomycin, or alkaline phosphatase, or protein- or peptide-based toxinemploys homobifunctional succinimidyl esters, preferably with carbonchain spacers such as disuccinimidyl suberate (Pierce Co, Rockford,Ill.). In the event that a cleavable conjugated compound is required,the same protocol would be employed utilizing 3,3′-dithiobis(sulfosuccinimidylpropionate; Pierce Co.).

In order to conjugate a GCC ligand that is a peptide or protein to apeptide-based active agent such as a toxin, the GCC ligand and the toxinmay be produced as a single, fusion protein either by standard peptidesynthesis or recombinant DNA technology, both of which can be routinelyperformed by those having ordinary skill in the art. Alternatively, twopeptides, the GCC ligand peptide and the peptide-based toxin may beproduced and/or isolated as separate peptides and conjugated usingcrosslinkers. As with conjugated compositions that containchemotherapeutic drugs, conjugation of GCC binding peptides and toxinscan exploit the ability to modify the single free amino group of a GCCbinding peptide while preserving the receptor-binding function of thismolecule.

One having ordinary skill in the art may conjugate a GCC ligand to aradionuclide using well-known techniques. For example, Magerstadt, M.(1991) Antibody Conjugates And Malignant Disease, CRC Press, Boca Raton,Fla.; and Barchel, S. W. and Rhodes, B. H., (1983) Radioimaging andRadiotherapy, Elsevier, NY, N.Y., each of which is incorporated hereinby reference, teach the conjugation of various therapeutic anddiagnostic radionuclides to amino acids of antibodies.

The present invention provides pharmaceutical compositions that comprisethe conjugated compounds of the invention and pharmaceuticallyacceptable carriers or diluents. The pharmaceutical composition of thepresent invention may be formulated by one having ordinary skill in theart. Suitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences, A. Osol, a standard reference text in thisfield, which is incorporated herein by reference. In carrying outmethods of the present invention, conjugated compounds of the presentinvention can be used alone or in combination with other diagnostic,therapeutic or additional agents. Such additional agents includeexcipients such as coloring, stabilizing agents, osmotic agents andantibacterial agents. Pharmaceutical compositions are preferably sterileand pyrogen free.

The conjugated compositions of the invention can be, for example,formulated as a solution, suspension or emulsion in association with apharmaceutically acceptable parenteral vehicle. Examples of suchvehicles are water, saline, Ringer's solution, dextrose solution, and 5%human serum albumin. Liposomes may also be used. The vehicle may containadditives that maintain isotonicity (e.g., sodium chloride, mannitol)and chemical stability (e.g., buffers and preservatives). Theformulation is sterilized by commonly used techniques. For example, aparenteral composition suitable for administration by injection isprepared by dissolving 1.5% by weight of active ingredient in 0.9%sodium chloride solution.

The pharmaceutical compositions according to the present invention maybe administered as either a single dose or in multiple doses. Thepharmaceutical compositions of the present invention may be administeredeither as individual therapeutic agents or in combination with othertherapeutic agents. The treatments of the present invention may becombined with conventional therapies, which may be administeredsequentially or simultaneously.

The pharmaceutical compositions of the present invention may beadministered by any means that enables the conjugated composition toreach the targeted cells. In some embodiments, routes of administrationinclude those selected from the group consisting of intravenous,intraarterial, intraperitoneal, local administration into the bloodsupply of the organ in which the tumor resides or directly into thetumor itself. Intravenous administration is the preferred mode ofadministration. It may be accomplished with the aid of an infusion pump.In addition to an intraoperative spray, conjuagated compounds may bedelivered intrathecally, intraventrically, stereotactically,intrahepatically such as via the portal vein, by inhalation, andintrapleurally.

The dosage administered varies depending upon factors such as: thenature of the active moiety; the nature of the conjugated composition;pharmacodynamic characteristics; its mode and route of administration;age, health, and weight of the recipient; nature and extent of symptoms;kind of concurrent treatment; and frequency of treatment.

Because conjugated compounds are specifically targeted to cells with oneor more GCC molecules, conjugated compounds which comprisechemotherapeutics or toxins are administered in doses less than thosewhich are used when the chemotherapeutics or toxins are administered asunconjugated active agents, preferably in doses that contain up to 100times less active agent. In some embodiments, conjugated compounds whichcomprise chemotherapeutics or toxins are administered in doses thatcontain 10-100 times less active agent as an active moiety than thedosage of chemotherapeutics or toxins administered as unconjugatedactive agents. To determine the appropriate dose, the amount of compoundis preferably measured in moles instead of by weight. In that way, thevariable weight of different GCC binding moieties does not affect thecalculation. Presuming a one to one ratio of GCC binding moiety toactive moiety in conjugated compositions of the invention, less moles ofconjugated compounds may be administered as compared to the moles ofunconjugated compounds administered, preferably up to 100 times lessmoles.

Typically, chemotherapeutic conjugates are administered intravenously inmultiple divided doses.

Up to 20 gm IV/dose of methotrexate is typically administered in anunconjugated form. When methotrexate is administered as the activemoiety in a conjugated compound of the invention, there is a 10- to100-fold dose reduction. Thus, presuming each conjugated compoundincludes one molecule of methotrexate conjugated to one GCC bindingmoiety, of the total amount of conjugated compound administered, up toabout 0.2-2.0 g of methotrexate is present and therefore administered.In some embodiments, of the total amount of conjugated compoundadministered, up to about 200 mg-2 g of methotrexate is present andtherefore administered.

To dose conjugated compositions comprising GCC binding moieties linkedto active moieties that are radioisotopes in pharmaceutical compositionsuseful as imaging agents, it is presumed that each GCC binding moiety islinked to one radioactive active moiety. The amount of radioisotope tobe administered is dependent upon the radioisotope. Those havingordinary skill in the art can readily formulate the amount of conjugatedcompound to be administered based upon the specific activity and energyof a given radionuclide used as an active moiety. Typically 0.1-100millicuries per dose of imaging agent, preferably 1-10 millicuries, mostoften 2-5 millicuries are administered. Thus, pharmaceuticalcompositions according to the present invention useful as imaging agentswhich comprise conjugated compositions comprising a GCC binding moietyand a radioactive moiety comprise 0.1-100 millicuries, in someembodiments preferably 1-10 millicuries, in some embodiments preferably2-5 millicuries, in some embodiments more preferably 1-5 millicuries.Examples of dosages include: ¹³¹I=between about 0.1-100 millicuries perdose, in some embodiments preferably 1-10 millicuries, in someembodiments 2-5 millicuries, and in some embodiments about 4millicuries; ¹¹¹In=between about 0.1-100 millicuries per dose, in someembodiments preferably 1-10 millicuries, in some embodiments 1-5millicuries, and in some embodiments about 2 millicuries;^(99m)Tc=between about 0.1-100 millicuries per dose, in some embodimentspreferably 5-75 millicuries, in some embodiments 10-50 millicuries, andin some embodiments about 27 millicuries. Wessels B. W. and R. D. Rogus(1984) Med. Phys. 11:638 and Kwok, C. S. et al. (1985) Med. Phys.12:405, both of which are incorporated herein by reference, disclosedetailed dose calculations for diagnostic and therapeutic conjugateswhich may be used in the preparation of pharmaceutical compositions ofthe present invention which include radioactive conjugated compounds.

One aspect of the present invention relates to a method of treatingindividuals suspected of suffering from primary and/or metastaticstomach or esophageal cancer. Such individuals may be treated byadministering to the individual a pharmaceutical composition thatcomprises a pharmaceutically acceptable carrier or diluent and aconjugated compound that comprises a GCC—binding moiety and an activemoiety wherein the active moiety is a radiostable therapeutic agent. Insome embodiments of the present invention, the pharmaceuticalcomposition comprises a pharmaceutically acceptable carrier or diluentand a conjugated compound that comprises a GCC binding moiety and anactive moiety wherein the active moiety is a radiostable active agentand the GCC binding moiety is an antibody. In some embodiments of thepresent invention, the pharmaceutical composition comprises apharmaceutically acceptable carrier or diluent and a conjugated compoundthat comprises a GCC binding moiety and an active moiety wherein theactive moiety is a radiostable therapeutic agent. In some embodiments ofthe present invention, the pharmaceutical composition comprises apharmaceutically acceptable carrier or diluent and a conjugated compoundthat comprises a GCC binding moiety and an active moiety wherein theactive moiety is a radiostable active agent selected from the groupconsisting of: methotrexate, doxorubicin, daunorubicin,cytosinarabinoside, etoposide, 5-4 fluorouracil, melphalan,chlorambucil, cis-platinum, vindesine, mitomycin, bleomycin,purothionin, macromomycin, 1,4-benzoquinone derivatives, trenimon,ricin, ricin A chain, Pseudomonas exotoxin, diphtheria toxin,Clostridium perfringens phospholipase C, bovine pancreatic ribonuclease,pokeweed antiviral protein, abrin, abrin A chain, cobra venom factor,gelonin, saporin, modeccin, viscumin, volkensin, alkaline phosphatase,nitroimidazole, metronidazole and misonidazole. The individual beingtreated may be diagnosed as having metastasized stomach or esophagealcancer or may be diagnosed as having primary stomach or esophagealcancer and may undergo the treatment proactively in the event that thereis some metastasis as yet undetected. The pharmaceutical compositioncontains a therapeutically effective amount of the conjugatedcomposition. A therapeutically effective amount is an amount which iseffective to cause a cytotoxic or cytostatic effect on cancer cellswithout causing lethal side effects on the individual.

One aspect of the present invention relates to a method of treatingindividuals suspected of suffering from primary and/or metastaticstomach or esophageal cancer. Such individuals may be treated byadministering to the individual a pharmaceutical composition thatcomprises a pharmaceutically acceptable carrier or diluent and aconjugated compound that comprises a GCC binding moiety and an activemoiety wherein the active moiety is a radioactive. In some embodimentsof the present invention, the pharmaceutical composition comprises apharmaceutically acceptable carrier or diluent and a conjugated compoundthat comprises a GCC binding moiety and an active moiety wherein theactive moiety is a radioactive and the GCC binding moiety is anantibody. In some embodiments of the present invention, thepharmaceutical composition comprises a pharmaceutically acceptablecarrier or diluent and a conjugated compound that comprises a GCCbinding moiety and an active moiety wherein the active moiety is aradioactive agent selected from the group consisting of: ⁴⁷Sc, ⁶⁷Cu,⁹⁰Y, ¹⁰⁹Pd, ¹²³I, ¹²⁵, ¹³¹I, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁹⁹Au, ²¹¹At, ²¹²Pb, ²¹²B,³²P and ³³P, ⁷¹Ge, ⁷⁷As, ¹⁰³Pb, ¹⁰⁵Rh, ¹¹¹Ag, ¹¹⁹Sb, ¹²¹Sn, ¹³¹Cs,¹⁴³Pr, ¹⁶¹Tb, ¹⁷⁷Lu, ¹⁹¹Os, ^(193M)Pt, ¹⁹⁷Hg, ³²P and ³³P, ⁷¹Ge, ⁷⁷As,¹⁰³Pb, ¹⁰⁵Rh, ¹¹¹Ag, ¹¹⁹Sb, ¹²¹Sn, ¹³¹Cs, ¹⁴³Pr, ¹⁶¹Tb, ¹⁷⁷Lu, ¹⁹¹Os,^(193M)Pt, ¹⁹⁷Hg, all beta negative and/or auger emitters. Theindividual being treated may be diagnosed as having metastasized canceror may be diagnosed as having localized cancer and may undergo thetreatment proactively in the event that there is some metastasis as yetundetected. The pharmaceutical composition contains a therapeuticallyeffective amount of the conjugated composition. A therapeuticallyeffective amount is an amount which is effective to cause a cytotoxic orcytostatic effect on primary and/or metastatic stomach or esophagealcancer cells without causing lethal side effects on the individual. Thecomposition may be injected intratumorally into primary tumors.

One aspect of the present invention relates to a method of detectingprimary and/or metastatic stomach or esophageal cancer cells in anindividual suspected of suffering from primary or metastasized stomachor esophageal cancer by radioimaging. Individuals may be suspected ofhaving primary stomach or esophageal tumors which diagnosis can beconfirmed by administering to the individual, an imaging agent whichbinds to GCC. Tumors can be imaged by detecting localization at thestomach or esophagus. Individuals may be diagnosed as suffering frommetastasized stomach or esophageal cancer and the metastasized stomachor esophageal cancer cells may be detected by administering to theindividual, preferably by intravenous administration, a pharmaceuticalcomposition that comprises a pharmaceutically acceptable carrier ordiluent and a conjugated compound that comprises a GCC binding moietyand an active moiety wherein the active moiety is a radioactive anddetecting the presence of a localized accumulation or aggregation ofradioactivity, indicating the presence of cells with GCC. In someembodiments of the present invention, the pharmaceutical compositioncomprises a pharmaceutically acceptable carrier or diluent and aconjugated compound that comprises a GCC binding moiety and an activemoiety wherein the active moiety is a radioactive and the GCC bindingmoiety is an antibody. In some embodiments of the present invention, thepharmaceutical composition comprises a pharmaceutically acceptablecarrier or diluent and a conjugated compound that comprises an GCCbinding moiety and an active moiety wherein the active moiety is aradioactive agent selected from the group consisting of: radioactiveheavy metals such as iron chelates, radioactive chelates of gadoliniumor manganese, positron emitters of oxygen, nitrogen, iron, carbon, orgallium, ⁴³K, ⁵²Fe, ⁵⁷Co, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁷Br, ⁸¹Rb/^(81M)Kr,^(87M)Sr, ^(99M)Tc, ¹¹¹In, ^(113M)In, ¹²³I, ¹²⁵I, ¹²⁷Cs, ¹²⁹Cs, ¹³¹I,¹³²I, ¹⁹⁷Hg, ²⁰³Pb and ²⁰⁶Bi. The individual being treated may bediagnosed as having metastasizing stomach or esophageal cancer or may bediagnosed as having localized stomach or esophageal cancer and mayundergo the treatment proactively in the event that there is somemetastasis as yet undetected. The pharmaceutical composition contains adiagnostically effective amount of the conjugated composition. Adiagnostically effective amount is an amount which can be detected at asite in the body where cells with GCC are located without causing lethalside effects on the individual.

Photodynamic Imaging and Therapy

According to some embodiments of the invention, GCC binding moieties areconjugates to photoactivated imaging agents or therapeutics. Maier A. etal. Lasers in Surgery and Medicine 26:461-466 (2000) which isincorporated herein by reference disclose an example of photodynamictherapy. QLT, Inc (Vancouver, BC) commercially distribute photosensitiveactive agents which can be linked to GCC ligands. Such conjugatedcompounds can be used in photodynamic therapeutic and imaging protocolsto activate the GCC-bound conjugated agents which are thus targeted totumor cells. In some embodiments, the conjugated compounds are appliedas an intraoperative spray which is subsequently exposed to light toactivate compounds bound to cells that express GCC.

In some embodiments, the photodynamic agent is fluorophore orporphyrins. Examples of porphyrin include: hematoporphyrin derivative(HPD) and porfimer sodium (Photofrin®). A second generationphotosensitizers is BPD verteporfin. In some embiodiments thefluorophore is tetramethylrotamine. Lasers are generally the primarylight source used to activate porphyrins. Light Emitting Diodes (LEDs)and florescent light sources may also be used in some applications.

In some embodiments, the photodynamic agent is linked to GCC at the GCCN-terminus.

In addition to an intraoperative spray, conjuagated compounds may bedelivered intrathecally, intraventrically, stereotactically,intrahepatically such as via the portal vein, by inhalation, andintrapleurally.

Drug Delivery Targeted to Stomach or Esophageal Cancer Cells Generally

Another aspect of the invention relates to unconjugated and conjugatedcompositions which comprise a GCC ligand used to deliver therapeuticagents to cells that comprise a GCC such as primary and/or metastaticstomach or esophageal cancer cells. In some embodiments, the agent is adrug or toxin such as: methotrexate, doxorubicin, daunorubicin,cytosinarabinoside, etoposide, 5-4 fluorouracil, melphalan,chlorambucil, cis-platinum, vindesine, mitomycin, bleomycin,purothionin, macromomycin, 1,4-benzoquinone derivatives, trenimon,ricin, ricin A chain, Pseudomonas exotoxin, diphtheria toxin,Clostridium perfringens phospholipase C, bovine pancreatic ribonuclease,pokeweed antiviral protein, abrin, abrin A chain, cobra venom factor,gelonin, saporin, modeccin, viscumin, volkensin, alkaline phosphatase,nitroimidazole, metronidazole and misonidazole. Genetic material isdelivered to cancer cells to produce an antigen that can be targeted bythe immune system or to produce a protein which kills the cell orinhibits its proliferation. In some embodiments, the GCC ligand is usedto deliver nucleic acids that encode nucleic acid molecules whichreplace defective endogenous genes or which encode therapeutic proteins.In some embodiments, the compositions are used in gene therapy protocolsto deliver to individuals, genetic material needed and/or desired tomake up for a genetic deficiency.

In some embodiments, the GCC ligand is combined with or incorporatedinto a delivery vehicle thereby converting the delivery vehicle into aspecifically targeted delivery vehicle. For example, a GCC bindingpeptide may be integrated into the outer portion of a viral particlemaking such a virus a GCC-bearing cell specific virus. Similarly, thecoat protein of a virus may be engineered such that it is produced as afusion protein which includes an active GCC binding peptide that isexposed or otherwise accessible on the outside of the viral particlemaking such a virus a GCC-bearing cell-specific virus. In someembodiments, a GCC ligand may be integrated or otherwise incorporatedinto the liposomes wherein the GCC ligand is exposed or otherwiseaccessible on the outside of the liposome making such liposomesspecifically targeted to GCC-bearing cells.

The active agent in the conjugated or unconjugated compositionsaccording to this aspect of the invention is a drug, toxin or nucleicacid molecule. The nucleic acid may be RNA or preferably DNA. In someembodiments, the nucleic acid molecule is an antisense molecule orencodes an antisense sequence whose presence in the cell inhibitsproduction of an undesirable protein. In some embodiments, the nucleicacid molecule encodes a ribozyme whose presence in the cell inhibitsproduction of an undesirable protein. In some embodiments, the nucleicacid molecule encodes a protein or peptide that is desirably produced inthe cell. In some embodiments, the nucleic acid molecule encodes afunctional copy of a gene that is defective in the targeted cell. Thenucleic acid molecule is preferably operably linked to regulatoryelements needed to express the coding sequence in the cell.

Liposomes are small vesicles composed of lipids. Genetic constructswhich encode proteins that are desired to be expressed in GCC-bearingcells are introduced into the center of these vesicles. The outer shellof these vesicles comprise an a GCC ligand. Liposomes Volumes 1, 2 and 3CRC Press Inc. Boca Raton Fla., which is incorporated herein byreference, disclose preparation of liposome-encapsulated active agentswhich include antibodies in the outer shell. In the present invention, aGCC ligand such as for example an anti-GCC antibodies is associated withthe in the outer shell. Unconjugated compositions which comprise a GCCligand in the matrix of a liposome with an active agent inside includesuch compositions in which the GCC ligand is preferably an antibody.

In one embodiment, the delivery of normal copies of the p53 tumorsuppressor gene to the cancer cells is accomplished using GCC ligand totarget the gene therapeutic. Mutations of the p53 tumor suppressor geneappears to play a prominent role in the development of many cancers. Oneapproach to combating this disease is the delivery of normal copies ofthis gene to the cancer cells expressing mutant forms of this gene.Genetic constructs that comprise normal p53 tumor suppressor genes areincorporated into liposomes that comprise a GCC ligand. The compositionis delivered to the tumor. GCC ligands specifically target and directthe liposomes containing the normal gene to correct the lesion createdby mutation of p53 suppressor gene. Preparation of genetic constructs iswith the skill of those having ordinary skill in the art. The presentinvention allows such construct to be specifically targeted by using theGCC ligands of the present invention. The compositions of the inventioninclude a GCC ligand such as an anti-GCC antibody associated with adelivery vehicle and a gene construct which comprises a coding sequencefor a protein whose production is desired in the cells of the intestinaltract linked to necessary regulatory sequences for expression in thecells. For uptake by cells of the intestinal tract, the compositions areadministered orally or by enema whereby they enter the intestinal tractand contact cells which comprise GCC. The delivery vehicles associatewith the GCC by virtue of the GCC ligand and the vehicle is internalizedinto the cell or the active agent/genetic construct is otherwise takenup by the cell. Once internalized, the construct can provide atherapeutic effect on the individual.

Antisense

The present invention provides compositions, kits and methods which areuseful to prevent and treat stomach or esophageal cancer cells byproviding the means to specifically deliver antisense compounds tostomach or esophageal cancer cells and thereby stop expression of genesin such cells in which undesirable gene expression is taking placewithout negatively effecting cells in which no such expression occurs.

The conjugated compositions of the present invention are useful fortargeting cells that express GCC including stomach or esophageal cancercells. The conjugated compositions will not bind to non-colorectalderived cells. Non-colorectal cells, lacking GCC, do not take up theconjugated compositions. Normal colorectal cells do have GCC and willtake up the compositions. The present invention provides compositionsand methods of delivering antisense compositions to stomach oresophageal cancer cells.

The present invention provides a cell specific approach in which onlynormal and cancerous colorectal cells and primary and/or metastaticstomach or esophageal cancer cells are exposed to the active portion ofthe compound and only those cells are effected by the conjugatedcompound. The GCC binding moiety binds to normal and cancerouscolorectal cells and primary and/or metastatic stomach or esophagealcancer cells. Upon binding to these cells, the conjugated compound isinternalized and the delivery of the conjugated compound including theantisense portion of the molecule is effected. The presence of theconjugated compound in normal colorectal cells has no effect on suchcells because the cancer-associated gene for which the antisensemolecule that makes up the active moiety of the conjugated compound iscomplementary is not being expressed. However, in colorectal cancercells, the cancer gene for which the antisense molecule that makes upthe active moiety of the conjugated compound is complementary is beingexpressed. The presence of the conjugated compound in colorectal cancercells serves to inhibit or prevent transcription or translation of thecancer gene and thereby reduce or eliminate the transformed phenotype.

The invention can be used to combat primary and/or metastasizedcolorectal, stomach or esophageal cancer as well as to prevent theemergence of the transformed phenotype in normal colon cells. Thus theinvention can be used therapeutically as well as prophylactically.

One having ordinary skill in the art can readily identify individualssuspected of suffering from stomach or esophageal cancer. In thoseindividuals diagnosed with stomach or esophageal cancer, it is standardtherapy to suspect metastasis and aggressively attempt to eradicatemetastasized cells. The present invention provides pharmaceuticalcompositions and methods for specifically targeting and eliminatingprimary and/or metastatic stomach or esophageal cancer cells. Further,the present invention provides pharmaceutical compositions that comprisetherapeutics and methods for specifically eliminating primary and/ormetastatic stomach or esophageal cancer cells.

The present invention relies upon the use of a GCC binding moiety in aconjugated composition. The GCC product binding moiety is essentially aportion of the conjugated composition which acts as a ligand to the GCCand thus specifically binds to these receptors. The conjugatedcomposition also includes an active moiety which is associated with theGCC binding moiety; the active moiety being an antisense compositionuseful to inhibit or prevent transcription or translation of expressionof genes whose expression is associated with cancer.

According to the present invention, the active moiety is an antisensecomposition. In particular, the antisense molecule that makes up theactive moiety of a conjugated compound hybridizes to DNA or RNA in astomach or esophageal cancer cell and inhibits and/or preventstranscription or translation of the DNA or RNA from taking place. Theantisense compositions may be a nucleic acid molecule, a derivative oran analogs thereof. The chemical nature of the antisense composition maybe that of a nucleic acid molecule or a modified nucleic acid moleculeor a non-nucleic acid molecule which possess functional groups thatmimic a DNA or RNA molecule that is complementary to the DNA or RNAmolecule whose expression is to be inhibited or otherwise prevented.Antisense compositions inhibit or prevent transcription or translationof genes whose expression is linked to stomach or esophageal cancer,i.e. cancer associated genes.

Point mutations insertions, and deletions in K-ras and H-ras have beenidentified in many tumors. Complex characteristics of the alterations ofoncogenes HER-2/ERBB-2, HER-1/ERBB-1, HRAS-1, C-MYC and anti-oncogenesp53, RB1.

Chemical carcinogenesis in a rat model demonstrated point mutations infos, an oncogene which mediates transcriptional regulation andproliferation. See: Alexander, R J, et al. Oncogene alterations in ratcolon tumors induced by N-methyl-N-nitrosourea. American Journal of theMedical Sciences. 303(1):16-24, 1992, January which is herebyincorporated herein by reference including all references cited thereinwhich are also hereby incorporated herein by reference.

Chemical carcinogenesis in a rat model demonstrated point mutations inthe oncogene abl. See: Alexander, R J, et al. Oncogene alterations inrat colon tumors induced by N-methyl-N-nitrosourea. American Journal ofthe Medical Sciences. 303(1): 16-24, 1992, January.

MYC is an oncogene that plays a role in regulating transcription andproliferation. A 15-base antisense oligonucleotide to myc complementaryto the translation initiation region of exon II was incubated withcolorectal cancer cells. This antisense molecule inhibited proliferationof colorectal cancer cells in a dos-dependent fashion. Interestingly,the uptake of this oligonucleotide was low (0.7%). Also, transfer of anormal chromosome 5 to colorectal cancer cells resulted in theregulation of myc expression and loss of proliferation. These datasuggest that a tumor suppressor gene important in the regulation of mycis contained on this chromosome.

A novel protein tyrosine phosphatase, G1, has been identified.Examination of the mRNA encoding this protein in colorectal tumor cellsrevealed that it undergoes point mutations and deletions in these cellsand may play a role in proliferation characteristic of these cells.Takekawa, M. et al. Chromosomal localization of the protein tyrosinephosphatase G1 gene and characterization of the aberrant transcripts inhuman colon cancer cells. FEBS Letters. 339(3):222-8, 1994 Feb. 21,which is hereby incorporated herein by reference including allreferences cited therein which are also hereby incorporated herein byreference.

Gastrin regulates colon cancer cell growth through a cyclicAMP-dependent mechanism mediated by PKA. Antisense oligodeoxynucleotidesto the regulatory subunit of a specific class of PKA inhibited thegrowth-promoting effects of cyclic AMP in colon carcinoma cells. See:Bold, R J, et al. Experimental gene therapy of human colon cancer.Surgery. 116(2):189-95; discussion 195-6, 1994 August and Yokozaki, H.,et al. An antisense oligodeoxynucleotide that depletes RI alpha subunitof cyclic AMP-dependent protein kinase induces growth inhibition inhuman cancer cells. Cancer Research. 53(4):868-72, 1993 Feb. 15, whichare both hereby incorporated herein by reference including allreferences cited therein which are also hereby incorporated herein byreference.

CRIPTO is an epidermal growth factor-related gene expressed in amajority of colorectal cancer tumors. Antisense phosphorothioateoligodeoxynucleotides to the 5′-end of CRIPTO mRNA significantly reducedCRIPTO expression and inhibited colorectal tumor cell growth in vitroand in vivo. Ciardiello, F. et al. Inhibition of CRIPTO expression andtumorigenicity in human colon cancer cells by antisense RNA andoligodeoxynucleotides. Oncogene. 9(1):291-8, 1994 January which are bothhereby incorporated herein by reference including all references citedtherein which are also hereby incorporated herein by reference.

Many carcinoma cells secrete transforming growth factor alpha. A 23nucleotide antisense oligonucleotide to TGF alpha mRNA inhibited bothDNA synthesis an proliferation of colorectal cancer cells. Sizeland, AM, Burgess, A W. Antisense transforming growth factor alphaoligonucleotides inhibit autocrine stimulated proliferation of a coloncarcinoma cell line. Molecular Biology of the Cell. 3(11):1235-43, 1992November which is hereby incorporated herein by reference including allreferences cited therein which are also hereby incorporated herein byreference.

Antisense compositions including oligonucleotides, derivatives andanalogs thereof, conjugation protocols, and antisense strategies forinhibition of transcription and translation are generally described in:Antisense Research and Applications, Crooke, S. and B. Lebleu, eds. CRCPress, Inc. Boca Raton Fla. 1993; Nucleic Acids in Chemistry and BiologyBlackburn, G. and M. J. Gait, eds. IRL Press at Oxford University Press,Inc. New York 1990; and Oligonucleotides and Analogues: A PracticalApproach Eckstein, F. ed., IRL Press at Oxford University Press, Inc.New York 1991; which are each hereby incorporated herein by referenceincluding all references cited therein which are hereby incorporatedherein by reference.

The antisense molecules of the present invention comprise a sequencecomplementary to a fragment of a colorectal cancer gene. See Ullrich etal., EMBO J., 1986, 5:2503, which is hereby incorporated herein byreference.

Antisense compositions which can make up an active moiety in conjugatedcompounds of the invention include oligonucleotides formed ofhomopyrimidines can recognize local stretches of homopurines in the DNAdouble helix and bind to them in the major groove to form a triplehelix. See: Helen, C and Toulme, J J. Specific regulation of geneexpression by antisense, sense, and antigene nucleic acids. Biochem.Biophys Acta, 1049:99-125, 1990 which is hereby incorporated herein byreference including all references cited therein which are herebyincorporated herein by reference. Formation of the triple helix wouldinterrupt the ability of the specific gene to undergo transcription byRNA polymerase. Triple helix formation using myc-specificoligonucleotides has been observed. See: Cooney, M, et al. Science241:456-459 which is hereby incorporated herein by reference includingall references cited therein which are hereby incorporated herein byreference.

Antisense oligonucleotides of DNA or RNA complementary to sequences atthe boundary between introns and exons can be employed to prevent thematuration of newly-generated nuclear RNA transcripts of specific genesinto mRNA for transcription.

Antisense RNA complimentary to specific genes can hybridize with themRNA for tat gene and prevent its translation. Antisense RNA can beprovided to the cell as “ready-to-use” RNA synthesized in vitro or as anantisense gene stably transfected into cells which will yield antisenseRNA upon transcription. Hybridization with mRNA results in degradationof the hybridized molecule by RNAse H and/or inhibition of the formationof translation complexes. Both result in a failure to produce theproduct of the original gene.

Antisense sequences of DNA or RNA can be delivered to cells. Severalchemical modifications have been developed to prolong the stability andimprove the function of these molecules without interfering in theirability to recognize specific sequences. These include increasing theirresistance to degradation by DNases, including phosphotriesters,methylphosphonates, phosphorothioates, alpha-anomers, increasing theiraffinity for their target by covalent linkage to various intercalatingagents such as psoralens, and increasing uptake by cells by conjugationto various groups including polylysine. These molecules recognizespecific sequences encoded in mRNA and their hybridization preventstranslation of and increases the degradation of these messages.

Conjugated compositions of the invention provide a specific andeffective means for terminating the expression of genes which causeneoplastic transformation. GCC undergo ligand-induced endocytosis andcan deliver conjugated compounds to the cytoplasm of cells.

GCC—binding moieties are conjugated directly to antisense compositionssuch as nucleic acids which are active in inducing a response. Forexample, antisense oligonucleotides to MYC are conjugated directly to ananti-GCC antibody. This has been performed employing peptides that bindto the CD4 receptor. See: Cohen, J S, ed. Oligodeoxynucleotides:Antisense Inhibitors of Gene Expression. Topics in Molecular andStructural Biology. CRC Press, Inc., Boca Raton, 1989. which is herebyincorporated herein by reference including all references cited thereinwhich are hereby incorporated herein by reference. The precise backboneand its synthesis is not specified and can be selected fromwell-established techniques. Synthesis would involve either chemicalconjugation or direct synthesis of the chimeric molecule by solid phasesynthesis employing FMOC chemistry. See: Haralambidis, J, et al. (1987)Tetrahedron Lett. 28:5199-5202, which is hereby incorporated herein byreference including all references cited therein which are herebyincorporated herein by reference. Alternatively, the peptide-nucleicacid conjugate may be synthesized directly by solid phase synthesis as apeptide-peptide nucleic acid chimera by solid phase synthesis. Nielsen,P E, et al. (1994) Sequence-specific transcription arrest by peptidenucleic acid bound to the DNA template strand. Gene 149:139-145, whichis hereby incorporated herein by reference including all referencescited therein which are hereby incorporated herein by reference.

In some embodiments, polylysine can be complexed to conjugatedcompositions of the invention in a non-covalent fashion to nucleic acidsand used to enhance delivery of these molecules to the cytoplasm ofcells. In addition, peptides and proteins can be conjugated topolylysine in a covalent fashion and this conjugate complexed withnucleic acids in a non-covalent fashion to further enhance thespecificity and efficiency of uptake of the nucleic acids into cells.Thus, GCC ligand is conjugated chemically to polylysine by establishedtechniques. The polylysine-GCC-1 translation product ligand conjugatemay be complexed with nucleic acids of choice. Thus,polylysine-orosomucoid conjugates were employed to specifically plasmidscontaining genes to be expressed to hepatoma cells expressing theorosomucoid receptor. This approach can be used to delivery whole genes,or oligonucleotides. Thus, it has the potential to terminate theexpression of an undesired gene (eg. MYC, ras) or replace the functionof a lost or deleted gene (eg. hMSH2, hMLH1, hPMS1, and hPMS2).

According to a preferred embodiment, Myc serves as a gene whoseexpression is inhibited by an antisense molecule within a conjugatedcomposition. GCC binding moieties are used to deliver a 15-basedantisense oligonucleotide to myc complementary to the translationinitiation region of exon II. The 15-base antisense oligonucleotide toMYC is synthesized as reported in Collins, J F, Herman, P, Schuch, C,Bagby GC, Jr. Journal of Clinical Investigation. 89(5):1523-7, 1992 May.In some embodiments, the conjugated composition is conjugated topolylysine as reported previously. Wu, G Y, and Wu, C H. (1988) Evidencefor ed gene delivery to Hep G2 hepatoma cells in vitro. Biochem.27:887-892 which is incorporated herein by reference.

Conjugated compositions may be synthesized as a chimeric moleculedirectly by solid phase synthesis. pmolar to nanomolar concentrationsfor this conjugate suppress MYC synthesis in colorectal cancer cells invitro.

Antisense molecules are preferably hybridize to, i.e. are complementaryto, a nucleotide sequence that is 5-50 nucleotides in length, morepreferably 5-25 nucleotides and in some embodiments 10-15 nucleotides.

In addition, mismatches within the sequences identified above, whichachieve the methods of the invention, such that the mismatched sequencesare substantially complementary to the cancer gene sequences are alsoconsidered within the scope of the disclosure. Mismatches which permitsubstantial complementarity to the cancer gene sequences will be knownto those of skill in the art once armed with the present disclosure. Theoligos may also be unmodified or modified.

Therapeutic compositions and methods may be used to combat stomach oresophageal cancer in cases where the cancer is localized and/ormetastasized. Individuals are administered a therapeutically effectiveamount of conjugated compound. A therapeutically effective amount is anamount which is effective to cause a cytotoxic or cytostatic effect oncancer cells without causing lethal side effects on the individual. Anindividual who has been administered a therapeutically effective amountof a conjugated composition has a increased chance of eliminatingstomach or esophageal cancer as compared to the risk had the individualnot received the therapeutically effective amount.

To treat localized stomach or esophageal cancer, a therapeuticallyeffective amount of a conjugated compound is administered such that itwill come into contact with the localized tumor. Thus, the conjugatedcompound may be administered orally or intratumorally. Oral and rectalformulation are taught in Remington's Pharmaceutical Sciences, 18thEdition, 1990, Mack Publishing Co., Easton Pa. which is incorporatedherein by reference.

The pharmaceutical compositions according to the present invention maybe administered as either a single dose or in multiple doses. Thepharmaceutical compositions of the present invention may be administeredeither as individual therapeutic agents or in combination with othertherapeutic agents. The treatments of the present invention may becombined with conventional therapies, which may be administeredsequentially or simultaneously.

The present invention is directed to a method of delivering antisensecompounds to normal and cancerous colorectal cells and to stomach oresophageal cancer cells and inhibiting expression of cancer genes inmammals. The methods comprise administering to a mammal an effectiveamount of a conjugated composition which comprises a GCC binding moietyconjugated to an antisense oligonucleotide having a sequence which iscomplementary to a region of DNA or mRNA of a cancer gene.

The conjugated compounds may be administering to mammals in a mixturewith a pharmaceutically-acceptable carrier, selected with regard to theintended route of administration and the standard pharmaceuticalpractice. Dosages will be set with regard to weight, and clinicalcondition of the patient. The conjugated compositions of the presentinvention will be administered for a time sufficient for the mammals tobe free of undifferentiated cells and/or cells having an abnormalphenotype. In therapeutic methods treatment extends for a timesufficient to inhibit transformed cells from proliferating andconjugated compositions may be administered in conjunction with otherchemotherapeutic agents to manage and combat the patient's cancer.

The conjugated compounds of the invention may be employed in the methodof the invention singly or in combination with other compounds. Theamount to be administered will also depend on such factors as the age,weight, and clinical condition of the patient. See Gennaro, Alfonso,ed., Remington's Pharmaceutical Sciences, 18th Edition, 1990, MackPublishing Co., Easton Pa.

Therapeutic and Prophylactic Vaccines

The invention relates to prophylactic and therapeutic vaccines forprotecting individuals against primary and/or metastatic stomach oresophageal cancer cells and for treating individuals who are sufferingfrom primary and/or metastatic stomach or esophageal cancer cells.

According to the present invention, GCC serves as targets against whicha protective and therapeutic immune response can be induced.Specifically, vaccines are provided which induce an immune responseagainst GCC. The vaccines of the invention include, but are not limitedto, the following vaccine technologies:

1) DNA vaccines, i.e. vaccines in which DNA that encodes at least anepitope from an GCC is administered to an individual's cells where theepitope is expressed and serves as a target for an immune response;

2) infectious vector mediated vaccines such as recombinant adenovirus,vaccinia, Salmonella, and BCG wherein the vector carries geneticinformation that encodes at least an epitope from an GCC protein suchthat when the infectious vector is administered to an individual, theepitope is expressed and serves as a target for an immune response;

3) killed or inactivated vaccines which a) comprise either killed cellsor inactivated viral particles that display at least an epitope from anGCC protein and b) when administered to an individual serves as a targetfor an immune response;

4) haptenized killed or inactivated vaccines which a) comprise eitherkilled cells or inactivated viral particles that display at least anepitope from an GCC protein, b) are haptenized to be more immunogenicand c) when administered to an individual serves as a target for animmune response;

5) subunit vaccines which are vaccines that include protein moleculesthat include at least an epitope from an GCC protein; and

6) haptenized subunit vaccines which are vaccines that a) includeprotein molecules that include at least an epitope from an GCC proteinand b) are haptenized to be more immunogenic.

The present invention relates to administering to an individual aprotein or nucleic acid molecule that comprises or encodes,respectively, an immunogenic epitope against which an therapeutic andprophylactic immune response can be induced. Such epitopes are generallyat least 6-8 amino acids in length. The vaccines of the inventiontherefore comprise proteins which are at least, or nucleic acids whichencode at least, 6-8 amino acids in length from GCC protein. Thevaccines of the invention may comprise proteins which are at least, ornucleic acids which encode at least 10 to about 1000 amino acids inlength. The vaccines of the invention may comprise proteins which are atleast, or nucleic acids which encode at least, about 25 to about 500amino acids in length. The vaccines of the invention may compriseproteins which are at least, or nucleic acids which encode at least,about 50 to about 400 amino acids in length. The vaccines of theinvention may comprise proteins which are at least, or nucleic acidswhich encode at least, about 100 to about 300 amino acids in length.

The present invention relates to compositions for and methods oftreating individuals who are known to have primary and/or metastaticstomach or esophageal cancer cells. Primary and/or metastatic stomach oresophageal cancer may be diagnosed by those having ordinary skill in theart using the methods described herein or art accepted clinical andlaboratory pathology protocols. The present invention provides animmunotherapeutic vaccine useful to treat individuals who have beendiagnosed as suffering from primary and/or metastatic stomach oresophageal cancer. The immunotherapeutic vaccines of the presentinvention may be administered in combination with other therapies.

The present invention relates to compositions for and methods ofpreventing primary and/or metastatic stomach or esophageal cancer inindividual is suspected of being susceptible to stomach or esophagealcancer. Such individuals include those whose family medical historyindicates above average incidence of stomach or esophageal cancer amongfamily members and/or those who have already developed stomach oresophageal cancer and have been effectively treated who therefore face arisk of relapse and recurrence. Such individuals include those whichhave been diagnosed as having stomach or esophageal cancer includinglocalized only or localized and metastasized stomach or esophagealcancer which has been resected or otherwise treated. The vaccines of thepresent invention may be to susceptible individuals prophylactically toprevent and combat primary and metastatic stomach or esophageal cancer.

The invention relates to compositions which are the active components ofsuch vaccines or required to make the active components, to methods ofmaking such compositions including the active components, and to methodsof making and using vaccines.

The amino acid and nucleotide sequences of the GCC is set forth as SEQID NO:1.

The present invention relates to recombinant vectors, includingexpression vectors, that comprise the GCC gene transcript or a fragmentthereof. The present invention relates to recombinant vectors, includingexpression vectors that comprise nucleotide sequences that encode a GCCprotein or a functional fragment thereof.

The present invention relates to host cells which comprise such vectorsand to methods of making GCC protein using such recombinant cells.

The present invention relates to the isolated GCC gene transcript and tothe isolated GCC proteins and to isolated antibodies specific for suchprotein and to hybridomas which produce such antibodies.

The present invention relates to the isolated GCC and functionalfragments thereof. Accordingly, some aspects of the invention relate toisolated proteins that comprise at least one epitope of an GCC.

Some aspects of the invention relate to the above described isolatedproteins which are haptenized to render them more immunogenic. That is,some aspects of the invention relate to haptenized proteins thatcomprise at least one GCC epitope.

Accordingly, some aspects of the invention relate to isolated nucleicacid molecules that encode proteins that comprise at least one GCCepitope.

Naked DNA vaccines are described in PCT/US90/01515, which isincorporated herein by reference. Others teach the use of liposomemediated DNA transfer, DNA delivery using microprojectiles (U.S. Pat.No. 4,945,050 issued Jul. 31, 1990 to Sanford et al., which isincorporated herein by reference), and DNA delivery usingelectroporation. In each case, the DNA may be plasmid DNA that isproduced in bacteria, isolated and administered to the animal to betreated. The plasmid DNA molecules are taken up by the cells of theanimal where the sequences that encode the protein of interest areexpressed. The protein thus produced provides a therapeutic orprophylactic effect on the animal.

The use of vectors including viral vectors and other means of deliveringnucleic acid molecules to cells of an individual in order to produce atherapeutic and/or prophylactic immunological effect on the individualare similarly well known.

Recombinant vaccines that employ vaccinia vectors are, for example,disclosed in U.S. Pat. No. 5,017,487 issued May 21, 1991 to Stunnenberget al. which is incorporated herein by reference.

In some cases, tumor cells from the patient are killed or inactivatedand administered as a vaccine product. Berd et al. May 1986 CancerResearch 46:2572-2577 and Berd et al. May 1991 Cancer Research51:2731-2734, which are incorporated herein by reference, describes thepreparation and use of tumor cell based vaccine products. According tosome aspects of the present invention, the methods and techniquesdescribed in Berd et al. are adapted by using stomach or esophagealcancer cells instead of melanoma cells.

The manufacture and use of isolated translation products and fragmentsthereof useful for example as laboratory reagents or components ofsubunit vaccines are well known. One having ordinary skill in the artcan isolate the GCC gene transcript or the specific portion thereof thatencodes GCC or a fragment thereof. Once isolated, the nucleic acidmolecule can be inserted it into an expression vector using standardtechniques and readily available starting materials.

The recombinant expression vector that comprises a nucleotide sequencethat encodes the nucleic acid molecule that encodes GCC or a fragmentthereof or a protein that comprises the GCC or a fragment thereof. Therecombinant expression vectors of the invention are useful fortransforming hosts to prepare recombinant expression systems forpreparing the isolated proteins of the invention.

The present invention relates to a host cell that comprises therecombinant expression vector that includes a nucleotide sequence thatencodes GCC protein or a fragment thereof or an GCC or a fragmentthereof. Host cells for use in well known recombinant expression systemsfor production of proteins are well known and readily available.Examples of host cells include bacteria cells such as E. coli, yeastcells such as S. cerevisiae, insect cells such as S. frugiperda,non-human mammalian tissue culture cells chinese hamster ovary (CHO)cells and human tissue culture cells such as HeLa cells.

The present invention relates to a transgenic non-human mammal thatcomprises the recombinant expression vector that comprises a nucleicacid sequence that encodes the proteins of the invention. Transgenicnon-human mammals useful to produce recombinant proteins are well knownas are the expression vectors necessary and the techniques forgenerating transgenic animals. Generally, the transgenic animalcomprises a recombinant expression vector in which the nucleotidesequence that encodes GCC or a fragment thereof or a protein thatcomprises GCC or a fragment thereof operably linked to a mammary cellspecific promoter whereby the coding sequence is only expressed inmammary cells and the recombinant protein so expressed is recovered fromthe animal's milk.

In some embodiments, for example, one having ordinary skill in the artcan, using well known techniques, insert such DNA molecules into acommercially available expression vector for use in well knownexpression systems such as those described herein.

The expression vector including the DNA that encodes a GCC or afunctional fragment thereof or a protein that comprises a GCC or afunctional fragment thereof is used to transform the compatible hostwhich is then cultured and maintained under conditions whereinexpression of the foreign DNA takes place. The protein of the presentinvention thus produced is recovered from the culture, either by lysingthe cells or from the culture medium as appropriate and known to thosein the art. The methods of purifying the GCC or a fragment thereof or aprotein that comprises the same using antibodies which specifically bindto the protein are well known. Antibodies which specifically bind to aparticular protein may be used to purify the protein from naturalsources using well known techniques and readily available startingmaterials. Such antibodies may also be used to purify the protein frommaterial present when producing the protein by recombinant DNAmethodology. The present invention relates to antibodies that bind to anepitope which is present on one or more GCC-1 translation products or afragment thereof or a protein that comprises the same. Antibodies thatbind to an epitope which is present on the GCC are useful to isolate andpurify the protein from both natural sources or recombinant expressionsystems using well known techniques such as affinity chromatography.Immunoaffinity techniques generally are described in Waldman et al. 1991Methods of Enzymol. 195:391-396, which is incorporated herein byreference. Antibodies are useful to detect the presence of such proteinin a sample and to determine if cells are expressing the protein. Theproduction of antibodies and the protein structures of complete, intactantibodies, Fab fragments and F(ab)₂ fragments and the organization ofthe genetic sequences that encode such molecules are well known and aredescribed, for example, in Harlow, E. and D. Lane (1988) ANTIBODIES: ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. which is incorporated herein by reference.

In some embodiments of the invention, transgenic non-human animals aregenerated. The transgenic animals according to the invention containnucleotides that encode GCC or a fragment thereof or a protein thatcomprises the same under the regulatory control of a mammary specificpromoter. One having ordinary skill in the art using standardtechniques, such as those taught in U.S. Pat. No. 4,873,191 issued Oct.10, 1989 to Wagner and U.S. Pat. No. 4,736,866 issued Apr. 12, 1988 toLeder, both of which are incorporated herein by reference, can producetransgenic animals which produce GCC or a fragment thereof or a proteinthat comprises the same. Preferred animals are goats and rodents,particularly rats and mice.

In addition to producing these proteins by recombinant techniques,automated peptide synthesizers may also be employed to produce GCC or afragment thereof or a fragment thereof or a protein that comprises thesame. Such techniques are well known to those having ordinary skill inthe art and are useful if derivatives which have substitutions notprovided for in DNA-encoded protein production.

In some embodiments, the protein that makes up a subunit vaccine or thecells or particles of a killed or inactivated vaccine may be haptenizedto increase immunogenicity. In some cases, the haptenization is theconjugation of a larger molecular structure to GCC or a fragment thereofor a protein that comprises the same. In some cases, tumor cells fromthe patient are killed and haptenized as a means to make an effectivevaccine product. In cases in which other cells, such as bacteria oreukaryotic cells which are provided with the genetic information to makeand display a GCC or a fragment thereof or a protein that comprises thesame, are killed and used as the active vaccine component, such cellsare haptenized to increase immunogenicity. Haptenization is well knownand can be readily performed.

Methods of haptenizing cells generally and tumor cells in particular aredescribed in Berd et al. May 1986 Cancer Research 46:2572-2577 and Berdet al. May 1991 Cancer Research 51:2731-2734, which are incorporatedherein by reference. Additional haptenization protocols are disclosed inMiller et al. 1976 J. Immunol. 117(5:1):1591-1526.

Haptenization compositions and methods which may be adapted to be usedto prepare haptenized GCC immunogens according to the present inventioninclude those described in the following U.S. patents which are eachincorporated herein by reference: U.S. Pat. No. 5,037,645 issued Aug. 6,1991 to Strahilevitz; U.S. Pat. No. 5,112,606 issued May 12, 1992 toShiosaka et al.; U.S. Pat. No. 4,526,716 issued Jul. 2, 1985 to Stevens;U.S. Pat. No. 4,329,281 issued May 11, 1982 to Christenson et al.; andU.S. Pat. No. 4,022,878 issued May 10, 1977 to Gross. Peptide vaccinesand methods of enhancing immunogenicity of peptides which may be adaptedto modify GCC immunogens of the invention are also described in Franciset al. 1989 Methods of Enzymol. 178:659-676, which is incorporatedherein by reference. Sad et al. 1992 Immunolology 76:599-603, which isincorporated herein by reference, teaches methods of makingimmunotherapeutic vaccines by conjugating gonadotropin releasing hormoneto diphtheria toxoid. GCC immunogens may be similarly conjugated toproduce an immunotherapeutic vaccine of the present invention. MacLeanet al. 1993 Cancer Immunol. Immunother. 36:215-222, which isincorporated herein by reference, describes conjugation methodologiesfor producing immunotherapeutic vaccines which may be adaptable toproduce an immunotherapeutic vaccine of the present invention. Thehapten is keyhole limpet hemocyanin which may be conjugated to a GCCimmunogen.

Vaccines according to some aspects of the invention comprise apharmaceutically acceptable carrier in combination with a GCC immunogen.Pharmaceutical formulations are well known and pharmaceuticalcompositions comprising such proteins may be routinely formulated by onehaving ordinary skill in the art. Suitable pharmaceutical carriers aredescribed in Remington's Pharmaceutical Sciences, A. Osol, a standardreference text in this field, which is incorporated herein by reference.The present invention relates to an injectable pharmaceuticalcomposition that comprises a pharmaceutically acceptable carrier and aGCC immunogen. The GCC immunogen is preferably sterile and combined witha sterile pharmaceutical carrier.

In some embodiments, for example, GCC or a fragment thereof or afragment thereof or a protein that comprises the same can be formulatedas a solution, suspension, emulsion or lyophilized powder in associationwith a pharmaceutically acceptable vehicle. Examples of such vehiclesare water, saline, Ringer's solution, dextrose solution, and 5% humanserum albumin. Liposomes and nonaqueous vehicles such as fixed oils mayalso be used. The vehicle or lyophilized powder may contain additivesthat maintain isotonicity (e.g., sodium chloride, mannitol) and chemicalstability (e.g., buffers and preservatives). The formulation issterilized by commonly used techniques.

An injectable composition may comprise the GCC immunogen in a dilutingagent such as, for example, sterile water, electrolytes/dextrose, fattyoils of vegetable origin, fatty esters, or polyols, such as propyleneglycol and polyethylene glycol. The injectable must be sterile and freeof pyrogens.

The vaccines of the present invention may be administered by any meansthat enables the immunogenic agent to be presented to the body's immunesystem for recognition and induction of an immunogenic response.Pharmaceutical compositions may be administered parenterally, i.e.,intravenous, subcutaneous, intramuscular.

Dosage varies depending upon known factors such as the pharmacodynamiccharacteristics of the particular agent, and its mode and route ofadministration; age, health, and weight of the recipient; nature andextent of symptoms, kind of concurrent treatment, frequency oftreatment, and the effect desired. An amount of immunogen is deliveredto induce a protective or therapeutically effective immune response.Those having ordinary skill in the art can readily determine the rangeand optimal dosage by routine methods.

The following examples are illustrative but are not meant to be limitingof the present invention.

EXAMPLES

As stated above, a GCC binding moiety is a GCC ligand that may be anantibody, a protein, a polypeptide, a peptide or a non-peptide. Peptidesand non-peptide GCC ligands may be identified using well knowntechnology.

Over the past 10 years, it has become recognized that the specifichigh-affinity interaction of a receptor and a ligand, for example a GCCand an anti-GCC antibody, has its basis in the 3-dimensionalconformational space of the ligand and the complimentary 3-dimensionalconfiguration of the region of the molecule involved in ligand binding.In addition, it has become recognized that various arrays ofnaturally-occurring amino acids, non-natural amino acids, and organicmolecules can be organized in configurations that are unrelated to thenatural ligands in their linear structure, but resemble the3-dimensional structure of the natural ligands in conformational spaceand, thus, are recognized by receptors with high affinity andspecificity. Furthermore, techniques have been described in theliterature that permit one of ordinary skill in the art to generatelarge libraries of these arrays of natural amino acids, non-naturalamino acids and organic compounds to prospectively identify individualcompounds that interact with receptors with high affinity andspecificity which are unrelated to the native ligand of that receptor.Thus, it is a relatively straightforward task for one of ordinary skillin the art to identify arrays of naturally occurring amino acids,non-natural amino acids, or organic compounds which can bindspecifically and tightly to the GCC, which bear no structuralrelationship to an anti-GCC antibody.

To identify GCC ligands that are peptides, those having ordinary skillin the art can use any of the well known methodologies for screeningrandom peptide libraries in order to identify peptides which bind to theGCC. In the most basic of methodologies, the peptides which bind to thetarget are isolated and sequenced. In some methodologies, each randompeptide is linked to a nucleic acid molecule which includes the codingsequence for that particular random peptide. The random peptides, eachwith an attached coding sequence, are contacted with a GCC and thepeptides which are unbound to the GCC are removed. The nucleic acidmolecule which includes the coding sequence of the peptide that binds tothe GCC can then be used to determine the amino acid sequence of thepeptide as well as produce large quantities of the peptide. It is alsopossible to produce peptide libraries on solid supports where thespatial location on the support corresponds to a specific synthesis andtherefore specific peptide. Such methods often use photolithography-likesteps to create diverse peptide libraries on solid supports in which thespatial address on the support allows for the determination of thesequence.

The production of organic compound libraries on solid supports may alsobe used to produce combinatorial libraries of non-peptide compounds suchas oligonucleotides and sugars, for example. As in the case of peptidelibraries on solid supports, the spatial location on the supportcorresponds to a specific synthesis and therefore specific compound.Such methods often use photolithography-like steps to create diversecompound libraries on solid supports in which the spatial address on thesupport allows for the determination of the synthesis scheme whichproduced the compound. Once the synthesis scheme is identified, thestructure of the compound can become known.

Gallop et al. 1994 J. Medicinal Chemistry 37:1233, which is incorporatedherein by reference, provides a review of several of the variousmethodologies of screening random peptide libraries and identifyingpeptides from such libraries which bind to target proteins. Followingthese teachings, GCC specific ligands that are peptides and that areuseful as GCC specific binding moieties may be identified by thosehaving ordinary skill in the art.

Peptides and proteins displayed on phage particles are described inGallop et al. Supra. Random arrays of nucleic acids can be inserted intogenes encoding surface proteins of bacteriophage which are employed toinfect bacteria, yielding phage expressing the peptides encoded by therandom array of nucleotides on their surface. These phage displaying thepeptide can be employed to determine whether those peptides can bind tospecific proteins, receptors, antibodies, etc. The identity of thepeptide can be determined by sequencing the recombinant DNA from thephage expressing the peptide. This approach has the potential to yieldvast arrays of peptides in a library (up to 10⁹ unique peptides). Thistechnique has been employed to identify novel binding peptides to thefibrinogen receptor on platelets, which bear no sequence homology to thenatural occurring ligands of this receptor (Smith et al., 1993 Gene128:37, which is incorporated herein by reference). Similarly, thistechnique has been applied to identify peptides which bind to the MHCclass II receptor (Hammer et al., 1993 Cell 74:197, which isincorporated herein by reference) and the chaperonin receptor(Blond-Elguindi et al., 1993 Cell 75:717, which is incorporated hereinby reference).

Peptides displayed on plasmids are described in Gallop et al. Supra. Inthis approach, the random oligonucleotides which encode the library ofpeptides can be expressed on a specific plasmid whose expression isunder the control of a specific promoter, such as the lac operon. Thepeptides are expressed as fusion proteins coupled to the Lac I protein,under the control of the lac operon. The fusion protein specificallybinds to the lac operator on the plasmid and so the random peptide isassociated with the specific DNA element that encodes it. In this way,the sequence of the peptide can be deduced, by PCR of the DNA associatedwith the fusion protein. These proteins can be screened in solutionphase to determine whether they bind to specific receptors. Employingthis approach, novel substrates have been identified for specificenzymes (Schatz 1993).

A variation of the above technique, also described in Gallop et al.Supra, can be employed in which random oligonucleotides encoding peptidelibraries on plasmids can be expressed in cell-free systems. In thisapproach, a molecular DNA library can be constructed containing therandom array of oligonucleotides, which are then expressed in abacterial in vitro transcription/translation system. The identity of theligand is determined by purifying the complex of nascent chainpeptide/polysome containing the mRNA of interest on affinity resinscomposed of the receptor and then sequencing following amplificationwith RT-PCR. Employing this technique permits generation of largelibraries (up to 10¹¹ recombinants). Peptides which recognize antibodiesspecifically directed to dynorphin have been identified employing thistechnique (Cull et al., 1992 Proc. Natl. Acad. Sci. USA 89:1865, whichis incorporated herein by reference).

Libraries of peptides can be generated for screening against a receptorby chemical synthesis. For example, simultaneous preparation of largenumbers of diverse peptides have been generated employing the approachof multiple peptide synthesis as described in Gallop et al. Supra. Inone application, random peptides are generated by standard solid-phaseMerrifield synthesis on polyacrylamide microtiter plates (multipinsynthesis) which are subsequently screened for their ability to competewith receptor binding in a standard competitive binding assay (Wang etal., 1993 Bioorg. Med. Chem. Lett. 3:447, which is incorporated hereinby reference). Indeed, this approach has been employed to identify novelbinding peptides to the substance P receptor (Wang et al. Supra).Similarly, peptide libraries can be constructed by multiple peptidesynthesis employing the “tea bag” method in which bags of solid supportresin are sequentially incubated with various amino acids to generatearrays of different peptides (Gallop et al. Supra). Employing thisapproach, peptides which bind to the integrin receptor (Ruggeri et al.,1986 Proc. Natl. Acad. Sci. USA 83:5708, which is incorporated herein byreference) and the neuropeptide Y receptor (Beck-Sickinger et al., 1990Int. J. Peptide Protein Res. 36:522, which is incorporated herein byreference) have been identified.

In general, the generation and utility of combinatorial libraries dependon (1) a method to generate diverse arrays of building blocks, (2) amethod for identifying members of the array that yield the desiredfunction, and (3) a method for deconvoluting the structure of thatmember. Several approaches to these constraints have been defined.

The following is a description of methods of library generation whichcan be used in procedures for identifying GCC ligands according to theinvention.

Modifications of the above approaches can be employed to generatelibraries of vast molecular diversity by connecting together members ofa set of chemical building blocks, such as amino acids, in all possiblecombinations (Gallop et al. Supra) In one approach, mixtures ofactivated monomers are coupled to a growing chain of amino acids on asolid support at each cycle. This is a multivalent synthetic system.

Also, split synthesis involves incubating the growing chain inindividual reactions containing only a single building block (Gallop etal. Supra). Following attachment, resin from all the reactions are mixedand apportioned into individual reactions for the next step of coupling.These approaches yield a stochastic collection of n^(x) differentpeptides for screening, where n is the number of building blocks and xis the number of cycles of reaction.

Alternatively, arrays of molecules can be generated in which one or morepositions contain known amino acids, while the remainder are random(Gallop et al. Supra). These yield a limited library which is screenedfor members with the desired activity. These members are identified,their structure determined, and the structure regenerated with anotherposition containing defined amino acids and screened. This iterativeapproach ultimately yields peptides which are optimal for recognizingthe conformational binding pocket of a receptor.

In addition, arrays are not limited to amino acids forming peptides, butcan be extended to linear and nonlinear arrays of organic molecules(Gordon et al., 1994 J. Medicinal Chemistry 37:1385, which isincorporated herein by reference). Indeed, employing this approach ofgenerating libraries of randomly arrayed inorganic building blocks,ligands which bound to 7-transmembrane receptors were identified(Zuckermann et al., 1994 J Med. Chem. 37:2678, which is incorporatedherein by reference).

Libraries are currently being constructed which can be modified aftersynthesis to alter the chemical side groups and bonds, to give“designer” arrays to test for their interaction with receptors (Ostereshet al., 1994 Proc. Natl. Acad. Sci. USA 91:11138, which is incorporatedherein by reference). This technique, generating “libraries fromlibraries”, was applied to the permethylation of a peptide library whichyielded compounds with selective antimicrobial activity against grampositive bacteria.

Libraries are also being constructed to express arrays ofpharmacological motifs, rather than specific structural arrays of aminoacids (Sepetov et al., 1995 Proc. Natl. Acad. Sci. USA 92:5426, which isincorporated herein by reference). This technique seeks to identifystructural motifs that have specific affinities for receptors, which canbe modified in further refinements employing libraries to definestructure-activity relationships. Employing this approach of searchingmotif libraries, generating “libraries of libraries”, reduces the numberof component members required for screening in the early phase oflibrary examination.

The following is a description of methods of identifying GCC ligandsaccording to the invention from libraries of randomly generatedmolecules.

Components in the library which interact with receptors may beidentified by their binding to receptors immobilized on solid support(Gordon et al. Supra).

They may also be identified by their ability to compete with nativeligand for binding to cognate receptors in solution phase (Gordon et al.Supra).

Components may be identified by their binding to soluble receptors whenthose components are immobilized on solid supports (Gordon et al.Supra).

Once a member of a library which binds receptors has been identified,the structure of that member must be deconvoluted (deduced) in order toidentify the structure and generate large quantities to work with, ordevelop further analogs to study structure-activity relationships. Thefollowing is a description of methods of deconvolution for deducing thestructure of molecules identified as potential GCC ligands according tothe invention.

Peptide libraries may be expressed on the surface of bacteriophageparticles (Gallop et al. Supra). Once the peptide interacting with thereceptor has been identified, its structure can be deduced by isolatingthe DNA from the phage and determining its sequence by PCR.

Libraries expressed on plasmids, under the control of the Lac operon canbe deconvoluted since these peptides are fused with the lac I proteinwhich specifically interacts with the lac operon on the plasmid encodingthe peptide (Gallop et al. Supra) The structure can be deduced byisolating that plasmid attached to the lac I protein and deducing thenucleotide and peptide sequence by PCR.

Libraries expressed on plasmids can also be expressed in cell-freesystems employing transcription/translation systems (Gallop et al.Supra). In this paradigm, the protein interacting with receptors isisolated with its attached ribosome and mRNA. The sequence of thepeptide is deduced by RT-PCR of the associated mRNA.

Library construction can be coupled with photolithography, so that thestructure of any member of the library can be deduced by determining itsposition within the substrate array (Gallop et al. Supra). Thistechnique is termed positional addressability, since the structuralinformation can be deduced by the precise position of the member.

Members of a library can also be identified by tagging the library withidentifiable arrays of other molecules (Ohlmeyer et al., 1993 Proc.Natl. Acad. Sci. USA 90:10922, which is incorporated herein byreference, and Gallop et al. Supra). This technique is a modification ofassociating the peptide with the plasmid of phage encoding the sequence,described above. Some methods employ arrays of nucleotides to encode thesequential synthetic history of the peptide. Thus, nucleotides areattached to the growing peptide sequentially, and can be decoded by PCRto yield the structure of the associated peptide. Alternatively, arraysof small organic molecules can be employed as sequencable tags whichencode the sequential synthetic history of the peptide. Thus,nucleotides are attached to the growing peptide sequentially, and can bedecoded by PCR to yield the structure of the associated peptide.Alternatively, arrays of small organic molecules can be employed assequencable tags which encode the sequential synthetic history of thelibrary member.

Finally, the structure of a member of the library can be directlydetermined by amino acid sequence analysis.

The following patents, which are each incorporated herein by reference,describe methods of making random peptide or non-peptide libraries andscreening such libraries to identify compounds that bind to targetproteins. As used in the present invention, GCC can be the targets usedto identify the peptide and non-peptide ligands generated and screenedas disclosed in the patents.

U.S. Pat. No. 5,270,170 issued to Schatz et al. on Dec. 14, 1993, andU.S. Pat. No. 5,338,665 issued to Schatz et al. on Aug. 16, 1994, whichare both incorporated herein by reference, refer to peptide librariesand screening methods which can be used to identify GCC ligands.

U.S. Pat. No. 5,395,750 issued to Dillon et al. on Mar. 7, 1995, whichis incorporated herein by reference, refers to methods of producingproteins which bind to predetermined antigens. Such methods can be usedto produce GCC ligands.

U.S. Pat. No. 5,223,409 issued to Ladner et al. on Jun. 29, 1993, whichis incorporated herein by reference, refers to the directed evolution tonovel binding proteins. Such proteins may be produced and screened asdisclosed therein to identify GCC ligands.

U.S. Pat. No. 5,366,862 issued to Venton et al. on Nov. 22, 1994, whichis incorporated herein by reference, refers to methods for generatingand screening useful peptides. The methods herein described can be usedto identify GCC ligands.

U.S. Pat. No. 5,340,474 issued to Kauvar on Aug. 23, 1994 as well asU.S. Pat. No. 5,133,866, U.S. Pat. No. 4,963,263 and U.S. Pat. No.5,217,869, which are each incorporated herein by reference, can be usedto identify GCC ligands.

U.S. Pat. No. 5,405,783 issued to Pirrung et al. on Apr. 11, 1995, whichis incorporated herein by reference, refers to large scalephotolithographic solid phase synthesis of an array of polymers. Theteachings therein can be used to identify GCC ligands.

U.S. Pat. No. 5,143,854 issued to Pirrung et al. on Sep. 1, 1992, whichis incorporated herein by reference, refers to a large scalephotolithographic solid phase synthesis of polypeptides and receptorbinding screening thereof.

U.S. Pat. No. 5,384,261 issued to Winkler et al. on Jan. 24, 1995, whichis incorporated herein by reference, refers to very large scaleimmobilized polymer synthesis using mechanically directed flow patterns.Such methods are useful to identify GCC ligands.

U.S. Pat. No. 5,221,736 issued to Coolidge et al. on Jun. 22, 1993,which is incorporated herein by reference, refers to sequential peptideand oligonucleotide synthesis using immunoaffinity techniques. Suchtechniques may be used to identify GCC ligands.

U.S. Pat. No. 5,412,087 issued to McGall et al. on May 2, 1995, which isincorporated herein by reference, refers to spatially addressableimmobilization of oligonucleotides and other biological polymers onsurfaces. Such methods may be used to identify GCC ligands.

U.S. Pat. No. 5,324,483 issued to Cody et al. on Jun. 28, 1994, which isincorporated herein by reference, refers to apparatus for multiplesimultaneous synthesis. The apparatus and method disclosed therein maybe used to produce multiple compounds which can be screened to identifyGCC ligands.

U.S. Pat. No. 5,252,743 issued to Barrett et al. on Oct. 12, 1993, whichis incorporated herein by reference, refers to spatially addressableimmobilization of anti-ligands on surfaces. The methods and compositionsdescribed therein may be used to identify GCC ligands.

U.S. Pat. No. 5,424,186 issued to Foder et al. on Jun. 13, 1995, whichis incorporated herein by reference, refers to a very large scaleimmobilized polymer synthesis. The method of synthesizingoligonucleotides described therein may be used to identify GCC ligands.

U.S. Pat. No. 5,420,328 issued to Campbell on May 30, 1995, which isincorporated herein by reference, refers to methods of synthesis ofphosphonate esters. The phosphonate esters so produced may be screenedto identify compounds which are GCC ligands.

U.S. Pat. No. 5,288,514 issued to Ellman on Feb. 22, 1994, which isincorporated herein by reference, refers to solid phase andcombinatorial synthesis of benzodiazepine compounds on a solid support.Such methods and compounds may be used to identify GCC ligands.

As noted above, GCC ligands may also be antibodies and fragmentsthereof. Indeed, antibodies raised to unique determinants of thesereceptors will recognize that protein, and only that protein and,consequently, can serve as a specific targeting molecule which can beused to direct novel diagnostics and therapeutics to this unique marker.In addition, these antibodies can be used to identify the presence ofGCC or fragments there of in biological samples.

1-33. (canceled)
 34. A method of treating an individual suspected ofsuffering from primary and/or metastatic stomach or esophageal cancercomprising the steps of administering to said individual atherapeutically effective amount of a composition comprising: i) aliposome; ii) an ST receptor ligand; and iii) an active agent. 35-38.(canceled)